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4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat-type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)-deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution. We found that similar to other TNFRSFs, m4-1BB has four cysteine-rich domains (CRDs). However, the organization of CRD1 and the orientation of CRD3 and CRD4 with respect to CRD2 in the m4-1BB structure distinctly differed from those of other TNFRSFs. Moreover, we mapped two Asn residues within CRD4 that are N -linked glycosylated and mediate m4-1BB binding to Gal-9. Kinetics studies of m4-1BB disclosed a very tight nanomolar binding affinity to m4-1BBL with an unexpectedly strong avidity effect. Both N- and C-terminal domains of Gal-9 bound m4-1BB, but with lower affinity compared with m4-1BBL. Although the TNF homology domain (THD) of human (h)4-1BBL forms non-covalent trimers, we found that m4-1BBL formed a covalent dimer via 2 cysteines absent in h4-1BBL. As multimerization and clustering is a prerequisite for TNFR intracellular signaling, and as m4-1BBL can only recruit two m4-1BB monomers, we hypothesize that m4-1BBL and Gal-9 act together to aid aggregation of m4-1BB monomers to efficiently initiate m4-1BB signaling., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: Several innate sensing pathways contribute to the control of early cytomegalovirus (CMV) infection, leading to a multiphasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells; however, the initial burst of IFN-I that occurs within the first few hours in vivo is TLR independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3) signaling. Disruption of STING expression in endothelial cells by clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 revealed that it is essential for the induction of IFN-I and restriction of CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in nonhematopoietic cells and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo ., Importance: Cytomegalovirus (CMV) is one of the most common viral pathogens, with the majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immunocompromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPRs revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice, STING is necessary for the first phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Since the discovery of TNF-related apoptosis-inducing ligand (TRAIL) and its network of receptors, the majority of attention has focused on the clinical potential of manipulating this pathway in cancer therapy. However, the widespread expression of TRAIL under inflammatory conditions and the ability to induce both apoptotic and prosurvival signaling pathways has suggested that TRAIL plays broader roles in regulating immune processes. Two new studies now show that expression of TRAIL by neutrophils in the lung facilitates defenses against bacterial pathogens, whereas expression of TRAIL by cells within arterioles exacerbates vascular disease. These differentiating results highlight that the context of TRAIL signaling can determine whether the outcome is beneficial or pathogenic for the host.

Type I interferons (IFN-αβ) are pleiotropic cytokines critical for antiviral host defense, and the timing and magnitude of their production involve a complex interplay between host and pathogen factors. Mouse cytomegalovirus (a β-herpesvirus) is a persistent virus that induces a biphasic IFN-αβ response during the first days of infection. The cell types and molecular mechanisms governing these 2 phases are unique, with splenic stromal cells being a major source of initial IFN-αβ, requiring communication with B cells expressing lymphotoxin, a tumor necrosis factor family cytokine. Here we review the factors that regulate this lymphotoxin-IFN-αβ "axis" during cytomegalovirus infection, highlight how stroma-derived IFN-αβ contributes in other models, and discuss how deregulation of this axis can lead to pathology in some settings.

The role of Cardif-dependent signaling in controlling dengue virus (DENV) infection and regulating type I interferon (IFN) production in vivo was examined in Cardif-deficient mice. DENV RNA levels were significantly elevated in both the serum and lymphoid tissues of Cardif(-/-) mice at early times compared to those in wild-type animals. Type I IFN production was delayed in these locales of Cardif(-/-) mice until 18 h postinfection, indicating that Cardif regulates the initial type I IFN response in lymphoid tissues. In contrast, DENV viral loads in nonlymphoid tissues were similar between Cardif(-/-) and wild-type mice. These results reveal that RNA helicase-mediated sensing acts as a first line of innate defense against DENV infection in vivo and functions in a tissue-dependent manner.

Early during infection, CMV targets dendritic cells (DC) and alters their functions. Herein we show that CMV-infected DC maintain the ability to present both virus-derived and exogenous Ags, but that they actively induce tolerance or anergy in Ag-specific T cells. CMV accomplishes this by selectively maintaining high-level expression of the negative costimulatory molecule programmed death ligand-1 (PD-L1), while commensurately down-regulating positive costimulatory molecules and MHC on the DC surface. Consequently, CD4 and CD8 T cells activated by these infected DC have a stunted phenotype, characterized by poor proliferation, effector function. and recall responses. Blocking PD-L1, but not PD-L2, during direct priming of naive T cells by infected DC significantly restores Ag-specific T cell functions. Using systems where direct and cross-priming of T cells can be distinguished revealed that PD-L1/PD-1 signaling contributes only when naive T cells are primed directly by infected DC, and not upon cross-presentation of viral Ags by uninfected DC. These data suggest that murine CMV programs infected DC during acute infection to inhibit early host adaptive antiviral responses by tipping the balance between negative and positive cosignals.

Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -epsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.

Lymphotoxin-beta receptor (LTbetaR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTbetaR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFkappaB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTbetaR truncated at position Pro(379) acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTbetaR. This mutant exhibited ligand-independent cell death and activated NF-kappaB p50 dimers. By contrast, truncation at Gly(359) created a dominant-negative mutant that inhibited ligand-induced cell death and activation of NF-kappaB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-kappaB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTbetaR.