The BTLA-HVEM complex - The future of cancer immunotherapy.

The BTLA-HVEM complex plays a pivotal role in cancer and cancer immunotherapy by regulating immune responses. Dysregulation of BTLA and HVEM expression contributes to immunosuppression and tumor progression across various cancer types. Targeting the interaction between BTLA and HVEM holds promise for enhancing anti-tumor immune responses. Disruption of this complex presents a valuable avenue for advancing cancer immunotherapy strategies. Aberrant expression of BTLA and HVEM adversely affects immune cell function, particularly T cells, exacerbating tumor evasion mechanisms. Understanding and modulating the BTLA-HVEM axis represents a crucial aspect of designing effective immunotherapeutic interventions against cancer. Here, we summarize the current knowledge regarding the structure and function of BTLA and HVEM, along with their interaction with each other and various immune partners. Moreover, the expression of soluble and transmembrane forms of BTLA and HVEM in different types of cancer and their impact on the prognosis of patients is also discussed. Additionally, inhibitors of the proteins binding that might be used to block BTLA-HVEM interaction are reviewed. All the presented data highlight the plausible clinical application of BTLA-HVEM targeted therapies in cancer and autoimmune disease management. However, further studies are required to confirm the practical use of this concept. Despite the increasing number of reports on the BTLA-HVEM complex, many aspects of its biology and function still need to be elucidated. This review can be regarded as an encouragement and a guide to follow the path of BTLA-HVEM research.

BTLA-derived peptides as inhibitors of BTLA/HVEM complex formation - design, synthesis and biological evaluation.

Immune checkpoints can be divided into co-stimulatory and co-inhibitory molecules that regulate the activation and effector functions of T cells. The co-inhibitory pathways mediated by ICPs are used by cancer cells to escape from immune surveillance, and therefore the blockade of these receptor/ligand interactions is one of the strategies used in the treatment of cancer. The two main pathways currently under investigation are CTLA-4/CD80/CD86 and PD-1/PD-L1, and the monoclonal Abs targeting them have shown potent immunomodulatory effects and activity in clinical environments. Another interesting target in cancer treatment is the BTLA/HVEM complex. Binding of BTLA protein on T cells to HVEM on cancer cells leads to inhibition of T cell proliferation and cytokine production. In the presented work, we focused on blocking the HVEM protein using BTLA-derived peptides. Based on the crystal structure of the BTLA/HVEM complex and MM/GBSA analysis performed here, we designed and synthesized peptides, specifically fragments of BTLA protein. We subsequently checked the inhibitory capacities of these compounds using ELISA and a cellular reporter platform. Two of these peptides, namely BTLA(35-43) and BTLA(33-64)C58Abu displayed the most promising properties, and we therefore performed further studies to evaluate their affinity to HVEM protein, their stability in plasma and their effect on viability of human PBMCs. In addition, the 3D structure for the peptide BTLA(33-64)C58Abu was determined using NMR. Obtained data confirmed that the BTLA-derived peptides could be the basis for future drugs and their immunomodulatory potential merits further examination.

BTLA inhibition has a dominant role in the cis-complex of BTLA and HVEM.

The engagement of the herpesvirus entry mediator (HVEM, TNFRSF14) by the B and T lymphocyte attenuator (BTLA) represents a unique interaction between an activating receptor of the TNFR-superfamily and an inhibitory receptor of the Ig-superfamily. BTLA and HVEM have both been implicated in the regulation of human T cell responses, but their role is complex and incompletely understood. Here, we have used T cell reporter systems to dissect the complex interplay of HVEM with BTLA and its additional ligands LIGHT and CD160. Co-expression with LIGHT or CD160, but not with BTLA, induced strong constitutive signaling via HVEM. In line with earlier reports, we observed that in cis interaction of BTLA and HVEM prevented HVEM co-stimulation by ligands on surrounding cells. Intriguingly, our data indicate that BTLA mediated inhibition is not impaired in this heterodimeric complex, suggesting a dominant role of BTLA co-inhibition. Stimulation of primary human T cells in presence of HVEM ligands indicated a weak costimulatory capacity of HVEM potentially owed to its in cis engagement by BTLA. Furthermore, experiments with T cell reporter cells and primary T cells demonstrate that HVEM antibodies can augment T cell responses by concomitantly acting as checkpoint inhibitors and co-stimulation agonists.

Targeting the HVEM protein using a fragment of glycoprotein D to inhibit formation of the BTLA/HVEM complex.

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein. To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10C-D30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD(1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

TNFSF14-Derived Molecules as a Novel Treatment for Obesity and Type 2 Diabetes.

Obesity is one of the most prevalent metabolic diseases in the Western world and correlates directly with glucose intolerance and insulin resistance, often culminating in Type 2 Diabetes (T2D). Importantly, our team has recently shown that the TNF superfamily (TNFSF) member protein, TNFSF14, has been reported to protect against high fat diet induced obesity and pre-diabetes. We hypothesized that mimics of TNFSF14 may therefore be valuable as anti-diabetic agents. In this study, we use in silico approaches to identify key regions of TNFSF14 responsible for binding to the Herpes virus entry mediator and Lymphotoxin ß receptor. In vitro evaluation of a selection of optimised peptides identified six potentially therapeutic TNFSF14 peptides. We report that these peptides increased insulin and fatty acid oxidation signalling in skeletal muscle cells. We then selected one of these promising peptides to determine the efficacy to promote metabolic benefits in vivo. Importantly, the TNFSF14 peptide 7 reduced high fat diet-induced glucose intolerance, insulin resistance and hyperinsulinemia in a mouse model of obesity. In addition, we highlight that the TNFSF14 peptide 7 resulted in a marked reduction in liver steatosis and a concomitant increase in phospho-AMPK signalling. We conclude that TNFSF14-derived molecules positively regulate glucose homeostasis and lipid metabolism and may therefore open a completely novel therapeutic pathway for treating obesity and T2D.

Redesigning HVEM Interface for Selective Binding to LIGHT, BTLA, and CD160.

Herpes virus entry mediator (HVEM) regulates positive and negative signals for T cell activation through co-signaling pathways. Dysfunction of the HVEM co-signaling network is associated with multiple pathologies related to autoimmunity, infectious disease, and cancer, making the associated molecules biologically and therapeutically attractive targets. HVEM interacts with three ligands from two different superfamilies using two different binding interfaces. The engagement with ligands CD160 and B- and T-lymphocyte attenuator (BTLA), members of immunoglobulin superfamily, is associated with inhibitory signals, whereas inflammatory responses are regulated through the interaction with LIGHT from the TNF superfamily. We computationally redesigned the HVEM recognition interfaces using a residue-specific pharmacophore approach, ProtLID, to achieve switchable-binding specificity. In subsequent cell-based binding assays the new interfaces, designed with only single or double mutations, exhibited selective binding to only one or two out of the three cognate ligands.

Disulfide-Linked Peptides for Blocking BTLA/HVEM Binding.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

Expression, purification, and efficient refolding of the extracellular domain of Escherichia coli-expressed signaling receptor herpesvirus entry mediator.

Herpesvirus entry mediator (HVEM), a member of the TNF-receptor superfamily, plays an important role in the regulation of the immune system. It forms a complex with ligands and can either activate or inhibit the response of the immune system. Furthermore, HVEM can exhibit pro-inflammatory or anti-inflammatory effects in many human diseases. Therefore, understanding the mechanism underlying the interaction of HVEM with other receptors is extremely important to design small therapeutic molecules that can stimulate the response of the immune system. In this study, we attempted to develop the most efficient method for the expression and purification of the extracellular domain of HVEM using Escherichia coli. The soluble fraction constituted only a small portion of the E. coli-expressed protein, whereas majority of the protein was found to be accumulated in the insoluble fraction. Three different protein refolding methods were analyzed: dialysis, dilution, and using chromatographic column. The oligomeric state of the protein was determined by characterizing the obtained fractions using analytical size exclusion chromatography. All the obtained fractions were tested for their ability to form a complex with B- and T-lymphocyte attenuator using enzyme-linked immunosorbent assay. The results of this study provide crucial information regarding the production of HVEM protein in a robust, well-established, and convenient heterologous expression system using E. coli as a host. In addition, it allows for the selection of the most effective method for appropriate refolding of HVEM protein, which gets accumulated in the insoluble fraction.

Structural Basis of CD160:HVEM Recognition.

CD160 is a signaling molecule that interacts with herpes virus entry mediator (HVEM) and contributes to a wide range of immune responses, including T cell inhibition, natural killer cell activation, and mucosal immunity. GPI-anchored and transmembrane isoforms of CD160 share the same ectodomain responsible for HVEM engagement, which leads to bidirectional signaling. Despite the importance of the CD160:HVEM signaling axis and its therapeutic relevance, the structural and mechanistic basis underlying CD160-HVEM engagement has not been described. We report the crystal structures of the human CD160 extracellular domain and its complex with human HVEM. CD160 adopts a unique variation of the immunoglobulin fold and exists as a monomer in solution. The CD160:HVEM assembly exhibits a 1:1 stoichiometry and a binding interface similar to that observed in the BTLA:HVEM complex. Our work reveals the chemical and physical determinants underlying CD160:HVEM recognition and initiation of associated signaling processes.

Structure of human cytomegalovirus UL144, an HVEM orthologue, bound to the B and T cell lymphocyte attenuator.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists.

A herpesvirus entry mediator mutein with selective agonist action for the inhibitory receptor B and T lymphocyte attenuator.

The human cytomegalovirus opening reading frame UL144 is an ortholog of the TNF receptor superfamily member, herpesvirus entry mediator (HVEM; TNFRSF14). HVEM binds the TNF ligands, LIGHT and LTa; the immunoglobulin inhibitory receptor, B and T lymphocyte attenuator (BTLA); and the natural killer cell-activating receptor CD160. However, UL144 selectively binds BTLA, avoiding activation of inflammatory signaling initiated by CD160 in natural killer cells. BTLA and CD160 cross-compete for binding HVEM, but the structural basis for the ligand selectivity by UL144 and how it acts as an anti-inflammatory agonist remains unclear. Here, we modeled the UL144 structure and characterized its binding with BTLA. The UL144 structure was predicted to closely mimic the surface of HVEM, and we also found that both HVEM and UL144 bind a common epitope of BTLA, whether engaged in trans or in cis, that is shared with a BTLA antibody agonist. On the basis of the UL144 selectivity, we engineered a BTLA-selective HVEM protein to understand the basis for ligand selectivity and BTLA agonism to develop novel anti-inflammatory agonists. This HVEM mutein did not bind CD160 or TNF ligands but did bind BTLA with 10-fold stronger affinity than wild-type HVEM and retained potent inhibitory activity that reduced T-cell receptor, B-cell receptor, and interferon signaling in B cells. In conclusion, using a viral immune evasion strategy that shows broad immune-ablating activity, we have identified a novel anti-inflammatory BTLA-selective agonist.

Design of short peptides to block BTLA/HVEM interactions for promoting anticancer T-cell responses.

Antibody based immune-checkpoint blockade therapy is a major breakthrough in oncology, leading to clinical benefit for cancer patients. Among the growing family of inhibitory receptors, the B and T lymphocyte attenuator (BTLA), which interacts with herpes virus entry mediator (HVEM), is a promising target for immunotherapy. Indeed, BTLA inhibits T-cell proliferation and cytokine production. The crystal structure of the BTLA/HVEM complex has shown that the HVEM(26-38) fragment is directly involved in protein binding. We designed and analyzed the capacity of several analogs of this fragment to block the ligation between BTLA and HVEM, using competitive ELISA and cellular assay. We found that the HVEM(23-39) peptide can block BTLA/HVEM ligation. However, the blocking ability was due to the Cys encompassed in this peptide and that even free cysteine targeted the BTLA protein and blocked its interaction with HVEM. These data highlight a Cys-related artefact in vitro, which should be taken in consideration for future development of BTLA/HVEM blocking compounds.

Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells.

The HVEM (TNFRSF14) receptor gene is among the most frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads to cell-autonomous activation of B cell proliferation and drives the development of GC lymphomas in vivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas in vivo, we engineered CD19-targeted chimeric antigen receptor (CAR) T cells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as "micro-pharmacies" able to deliver an anti-cancer protein.

Increased Heterologous Protein Expression in Drosophila S2 Cells for Massive Production of Immune Ligands/Receptors and Structural Analysis of Human HVEM.

Many immune ligands and receptors are potential drug targets, which delicately manipulate a wide range of immune responses. We describe here the successful application of an efficient method to dramatically improve the heterologous expression levels in Drosophila Schneider 2 cells, which enables the high-throughput production of several important immune ligands/receptors for raising antibodies, and for the structural and functional analyses. As an example, we purified the protein and characterized the structure of the immune receptor herpesvirus entry mediator (HVEM, TNFRSF14). HVEM is a member of tumor necrosis factor receptor superfamily, which is recognized by herpes simplex virus glycoprotein D (gD) and facilitates viral entry. HVEM participates in a range of interactions with other cell surface molecules, including LIGHT, BTLA, and CD160 to modulate a wide range of immune processes in CD4(+) and CD8(+) T cells, as well as NK cells. Due to the involvement of HVEM in these diverse signaling interactions, crystal structures of HVEM in complex with gD or BTLA have been previously reported. Here, we report the structure of HVEM in the absence of any ligands.

Functional characterization of viral tumor necrosis factor receptors encoded by cyprinid herpesvirus 3 (CyHV3) genome.

Cyprinid herpesvirus 3 (CyHV3) is a large double-stranded DNA virus of Alloherpesviridae family in the order Herpesvirales. It causes significant morbidity and mortality in common carp and its ornamental koi variety, and threatens the aquaculture industries worldwide. Mimicry of cytokines and cytokine receptors is a particular strategy for large DNA viruses in modulating the host immune response. Here, we report the identification and characterization of two novel viral homologues of tumor necrosis factor receptor (TNFR) encoded by CyHV3-ORF4 and -ORF12, respectively. CyHV3-ORF4 was identified as a homologue of HVEM and CyHV3-ORF12 as a homologue of TNFRSF1. Overexpression of ORF4 and ORF12 in zebrafish embryos results in embryonic lethality, morphological defects and increased apoptosis. Although we failed to identify any interaction between the two vTNFRs and their potential ligands in zebrafish TNF superfamily by yeast two-hybrid system, the expression of some genes in TNF superfamily or TNFR superfamily were mis-regulated in ORF4 or ORF12-overexpressing embryos, especially the death receptor zHDR and its cognate ligand DL1b. Further studies showed that the apoptosis induced by the both CyHV3 vTNFRs is mainly activated through the intrinsic apoptotic pathway and requires the crosstalk between the intrinsic and extrinsic apoptotic pathway. Additionally, using RT-qPCR and Western blot assays, the expression patterns of the both vTNFRs were also analyzed during CyHV3 productive infection. Collectively, this is the first functional study of two unique vTNFRs encoded by a herpesvirus infecting non-mammalian vertebrates, which may provide novel insights into viral immune regulation mechanism and the pathogenesis of CyHV3 infection.

Molecular cloning and characterization of TNFSF14 (LIGHT) and its receptor TNFRSF14 (HVEM) in guinea pig (Cavia porcellus).

LIGHT (lymphotoxin-related inducible ligand that competes with herpes simplex virus (HSV) glycoprotein D for herpesvirus entry mediator on T cells) is a member of the tumor necrosis factor (TNF) ligand superfamily, which plays important roles in inflammatory and immune responses. In the present study, the cDNAs of guinea pig (Cavia porcellus) LIGHT (designated as gpLIGHT) and its receptor herpes virus entry mediator (designated as gpHVEM) were amplified from spleen by reverse transcription polymerase chain reaction (RT-PCR). The ORFs of gpLIGHT and gpHVEM cover 726 and 861 bp, encoding predicted proteins with 241 and 286 aas, respectively. The three-dimensional (3D) structure, phylogenetic relationships, and characterization of both genes were also analyzed. We also generated a 3D model to verify interaction between the two proteins. Real-time quantitative PCR (qPCR) analysis revealed that both LIGHT and HVEM are constitutively expressed in guinea pig various tissues. A fusion protein SUMO (Small Ubiquitin-like Modifier)-gpsLIGHT (the soluble mature part of gpLIGHT) was efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy (LSCM) showed that gpsLIGHT can bind its receptors on T cells. The LIGHT-HVEM signaling pathway plays an important role in the immune system, and our results might provide a platform for further research into the effects of LIGHT and HVEM.

Molecular basis for herpesvirus entry mediator recognition by the human immune inhibitory receptor CD160 and its relationship to the cosignaling molecules BTLA and LIGHT.

CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4(+) T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function.

Herpes simplex virus glycoprotein D interferes with binding of herpesvirus entry mediator to its ligands through downregulation and direct competition.

To initiate membrane fusion and virus entry, herpes simplex virus (HSV) gD binds to a cellular receptor such as herpesvirus entry mediator (HVEM). HVEM is a tumor necrosis factor (TNF) receptor family member with four natural ligands that either stimulate (LIGHT and LTα) or inhibit (BTLA and CD160) T cell function. We hypothesized that the interaction of gD with HVEM affects the binding of natural ligands, thereby modulating the immune response during infection. Here, we investigated the effect that gD has on the interaction of HVEM with its natural ligands. First, HSV gD on virions or cells downregulates HVEM from the cell surface. Similarly, trans-interaction with BTLA or LIGHT also downregulates HVEM from the cell surface, suggesting that HSV may subvert a natural mechanism for regulating HVEM activity. Second, we showed that wild-type gD had the lowest affinity for HVEM compared with the four natural ligands. Moreover, gD directly competed for binding to HVEM with BTLA but not LTα or LIGHT, indicating the possibility that gD selectively controls HVEM signals. On the other hand, natural ligands influence the use of HVEM by HSV. For instance, soluble BTLA, LTα, and LIGHT inhibited the binding of wild-type gD to HVEM, and soluble BTLA and LTα blocked HSV infection of HVEM-expressing cells. Thus, gD is at the center of the interplay between HVEM and its ligands. It can interfere with HVEM function in two ways, by competing with the natural ligands and by downregulating HVEM from the cell surface.

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-kappaB RelA transcriptional complex raising the question of how NF-kappaB 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-kappaB 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-kappaB 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-kappaB 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-kappaB 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.