Timothy C. Cheung

LIGHT, a New Member of the TNF Superfamily, and Lymphotoxin α Are Ligands for Herpesvirus Entry Mediator

Herpes simplex virus (HSV) 1 and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D (gD) to the cellular herpesvirus entry mediator (HVEM), an orphan member of the tumor necrosis factor receptor superfamily. Here, we demonstrate that HVEM binds two cellular ligands, secreted lymphotoxin alpha (LTalpha) and LIGHT, a new member of the TNF superfamily. LIGHT is a 29 kDa type II transmembrane protein produced by activated T cells that also engages the receptor for the LTalphabeta heterotrimer but does not form complexes with either LTalpha or LTbeta. HSV1 gD inhibits the interaction of HVEM with LIGHT, and LIGHT and gD interfere with HVEM-dependent cell entry by HSV1. This characterizes herpesvirus gD as a membrane-bound viokine and establishes LIGHT-HVEM as integral components of the lymphotoxin cytokine-receptor system.

Unconventional ligand activation of herpesvirus entry mediator signals cell survival

The herpesvirus entry mediator (HVEM; TNFRSF14) activates NF-κB through the canonical TNF-related cytokine LIGHT, serving as a costimulatory pathway during activation of T cells. HVEM also functions as a ligand for the Ig superfamily members B and T lymphocyte attenuator (BTLA) and CD160, both of which limit inflammatory responses initiated by T cells. Emerging evidence indicates BTLA also promotes T cell survival, but its structural differences from LIGHT intimate BTLA is unlikely to function as an activator of HVEM. We demonstrate here that BTLA, CD160, and herpes simplex virus envelope glycoprotein D (gD) function as activating ligands for HVEM, promoting NF-κB activation and cell survival. Membrane-expressed BTLA and CD160, as well as soluble dimeric receptor surrogates BTLA-Fc and gD-Fc specifically activated HVEM-dependent NF-κB. BTLA and CD160 engagement induced recruitment of TNF receptor-associated factor 2 (TRAF2), but not TRAF3, to HVEM that specifically activated the RelA but not the RelB form of NF-κB in a mucosal epithelial tumor cell line. Moreover, Btla−/− T cells survived poorly following activation but were rescued with BTLA-Fc, indicating HVEM-BTLA bidirectional signaling may serve as a critical cell-survival system for lymphoid and epithelial cells.

Dominant Negative Mutants of TRAF3 Reveal an Important Role for the Coiled Coil Domains in Cell Death Signaling by the Lymphotoxin-β Receptor

Ligation of the lymphotoxin-β receptor (LTβR) recruits tumor necrosis factor receptor-associated factor-3 (TRAF3) and initiates cell death in HT29 adenocarcinoma cells. The minimal receptor binding domain (TRAF-C) defined by two hybrid analyses is not sufficient for direct recruitment to the ligated receptor. A series of TRAF3 deletion mutants reveal that a subregion of the coiled coil motif is required for efficient recruitment to the LTβR. Furthermore, the ability of TRAF3 to self-associate maps to an adjacent subregion. A TRAF3 deletion mutant that lacks the N-terminal zinc RING and zinc finger motifs, but retains the coiled coil and TRAF-C motifs, competitively displaces endogenous TRAF3 from the LTβR. A second TRAF3 mutant that lacks the receptor binding domain, yet contains the TRAF3 self-association domain, prevents TRAF3 homodimers from being recruited to the LTβR. Both of these mutants have a dominant negative effect on cell death and demonstrate that the recruitment of TRAF3 oligomers is necessary to initiate signal transduction that activates the cell death pathway.

The Signaling Networks of the Herpesvirus Entry Mediator (TNFRSF14) in Immune Regulation

Summary:u2002 The tumor necrosis factor (TNF) receptor superfamily member herpesvirus entry mediator (HVEM) (TNFRSF14) regulates T‐cell immune responses by activating both inflammatory and inhibitory signaling pathways. HVEM acts as both a receptor for the canonical TNF‐related ligands, LIGHT [lymphotoxin‐like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed on T lymphocytes] and lymphotoxin‐α, and as a ligand for the immunoglobulin superfamily proteins BTLA (B and T lymphocyte attenuator) and CD160, a feature distinguishing HVEM from other immune regulatory molecules. The ability of HVEM to interact with multiple ligands in distinct configurations creates a functionally diverse set of intrinsic and bidirectional signaling pathways that control both inflammatory and inhibitory responses. The HVEM system is integrated into the larger LTβR and TNFR network through extensive shared ligand and receptor usage. Experimental mouse models and human diseases indicate that dysregulation of HVEM network may contribute to autoimmune pathogenesis, making it an attractive target for drug intervention.

T Cell Intrinsic Heterodimeric Complexes between HVEM and BTLA Determine Receptivity to the Surrounding Microenvironment

The inhibitory cosignaling pathway formed between the TNF receptor herpesvirus entry mediator (HVEM, TNFRSF14) and the Ig superfamily members, B and T lymphocyte attenuator (BTLA) and CD160, limits the activation of T cells. However, BTLA and CD160 can also serve as activating ligands for HVEM when presented in trans by adjacent cells, thus forming a bidirectional signaling pathway. BTLA and CD160 can directly activate the HVEM-dependent NF-κB RelA transcriptional complex raising the question of how NF-κB activation is repressed in naive T cells. In this study, we show BTLA interacts with HVEM in cis, forming a heterodimeric complex in naive T cells that inhibits HVEM-dependent NF-κB activation. The cis-interaction between HVEM and BTLA is the predominant form expressed on the surface of naive human and mouse T cells. The BTLA ectodomain acts as a competitive inhibitor blocking BTLA and CD160 from binding in trans to HVEM and initiating NF-κB activation. The TNF-related ligand, LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14) binds HVEM in the cis-complex, but NF-κB activation was attenuated, suggesting BTLA prevents oligomerization of HVEM in the cis-complex. Genetic deletion of BTLA or pharmacologic disruption of the HVEM-BTLA cis-complex in T cells promoted HVEM activation in trans. Interestingly, herpes simplex virus envelope glycoprotein D formed a cis-complex with HVEM, yet surprisingly, promoted the activation NF-κB RelA. We suggest that the HVEM-BTLA cis-complex competitively inhibits HVEM activation by ligands expressed in the surrounding microenvironment, thus helping maintain T cells in the naive state.

The lymphotoxin-β receptor is an upstream activator of NF-κB-mediated transcription in melanoma cells

The pleiotropic transcription factor nuclear factor-κB (NF-κB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-κB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-κB has not been elucidated in the tumors. We have previously shown that NF-κB-inducing kinase (NIK) protein and its association with Inhibitor of κB kinase αβ are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-κB. Moreover, expression of dominant negative NIK blocked this base-line NF-κB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-κB, only the lymphotoxin-β receptor (LTβ-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTβ-R and constitutive NF-κB transcriptional activity. Moreover, we show that activation of the LTβ-R can drive NF-κB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTβ-R shRNA resulted in decreased NF-κB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTβ-R constitutively induces NF-κB activation, and this event may be associated with autonomous growth of melanoma cells.

The Lymphotoxin-α (LTα) Subunit Is Essential for the Assembly, but Not for the Receptor Specificity, of the Membrane-anchored LTα1β2 Heterotrimeric Ligand

The lymphotoxins (LT) α and β, members of the tumor necrosis factor (TNF) cytokine superfamily, are implicated as important regulators and developmental factors for the immune system. LTα is secreted as a homotrimer and signals through two TNF receptors of 55–60 kDa (TNFR60) or 75–80 kDa (TNFR80). LTα also assembles with LTβ into a membrane-anchored, heterotrimeric LTα1β2 complex that engages a distinct cognate receptor, the LTβ receptor (LTβR). To investigate the role of the LTα subunit in the function of the membrane LTα1β2 complex, gene transfer via baculovirus was used to assemble LTα and -β complexes in insect cells. LTα containing mutations at D50N or Y108F are secreted as homotrimers that fail to bind either TNF receptor and are functionally inactive in triggering cell death of the HT29 adenocarcinoma cell line. In contrast, these mutant LTα proteins retain the ability to co-assemble with LTβ into membrane-anchored LTα1β2 complexes that engage the LTβR and trigger the death of HT29 cells. Membrane-anchored LTβ expressed on the cell surface in absence of the LTα subunit binds the LTβR but is functionally inactive in the cell death assay. These results indicate that the TNF receptor-binding regions of the LTα subunit are not necessary for engagement of the LTβR, but the LTα subunit is required for the assembly of LTβ into a functional heteromeric ligand.

Polymorphic Variants of LIGHT (TNF Superfamily-14) Alter Receptor Avidity and Bioavailability

The TNF superfamily member homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes (LIGHT) [TNF superfamily (SF)-14], is a key cytokine that activates T cells and dendritic cells and is implicated as a mediator of inflammatory, metabolic, and malignant diseases. LIGHT engages the lymphotoxin-β receptor (LTβR) and HVEM (TNFRSF14), but is competitively limited in activating these receptors by soluble decoy receptor-3 (DcR3; TNFRSF6B). Two variants in the human LIGHT alter the protein at E214K (rs344560) in the receptor-binding domain and S32L (rs2291667) in the cytosolic domain; however, the functional impact of these polymorphisms is unknown. A neutralizing Ab failed to bind the LIGHT-214K variant, indicating this position as a part of the receptor-binding region. Relative to the predominant reference variant S32/E214, the other variants showed altered avidity with LTβR and less with HVEM. Heterotrimers of the LIGHT variants decreased binding avidity to DcR3 and minimized the inhibitory effect of DcR3 toward LTβR-induced activation of NF-κB. In patients with immune-mediated inflammatory diseases, such as rheumatoid arthritis, DcR3 protein levels were significantly elevated. Immunohistochemistry revealed synoviocytes as a significant source of DcR3 production, and DcR3 hyperexpression is controlled by posttranscriptional mechanisms. The increased potential for LTβR signaling, coupled with increased bioavailability due to lower DcR3 avidity, provides a mechanism of how polymorphic variants in LIGHT could contribute to the pathogenesis of inflammatory diseases.

The canonical and unconventional ligands of the herpesvirus entry mediator.

The maintenance of immune homeostasis requires a balance between stimulatory and inhibitory pathways. The herpesvirus entry mediator (HVEM; TNFRSF14) [1] serves as one of two key entry routes used by herpes simplex virus-1 and herpes simplex virus-2 to infect cells [2].