Epitope topography of agonist antibodies to the checkpoint inhibitory receptor BTLA.

B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.

The signaling networks of the herpesvirus entry mediator (TNFRSF14) in immune regulation.

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 LTßR 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.

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-beta receptor (LTbetaR) 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 LTbetaR and less with HVEM. Heterotrimers of the LIGHT variants decreased binding avidity to DcR3 and minimized the inhibitory effect of DcR3 toward LTbetaR-induced activation of NF-kappaB. 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 LTbetaR 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.

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.

Modulation of T cell proliferation through the LIGHT-HVEM-BTLA cosignaling pathway.

The maintenance of T cell homeostasis requires the balance of both positive and negative regulatory signals of T cell proliferation. The herpesvirus entry mediator (HVEM) is a TNF superfamily receptor member, which provides a stimulatory signal following engagement with LIGHT (TNFSF14) on T cells. In contrast, HVEM can also provide an inhibitory signal to T cells when it binds the B and T lymphocyte attenuator (BTLA), a ligand member of the Immunoglobulin (Ig) superfamily. Thus, HVEM may be viewed as a molecular switch, capable of facilitating both stimulatory and inhibitory cosignaling in T cells. Substantial evidence from both human disease and from experimental mouse models has indicated that dysregulation of the LIGHT-HVEM-BTLA cosignaling pathway can cause inflammation in the lung and in mucosal tissues. Moreover, the discovery that two different viruses, human cytomegalovirus (HCMV) and herpes simplex virus (HSV) both target the LIGHT-HVEM-BTLA system as part of their immune evasion strategies suggests that this cosignaling pathway may prove useful in manipulating host immunity. Indeed, the LIGHT-HVEM-BTLA system may constitute an important molecular target for biopharmaceutical intervention as it holds the key for controlling both costimulatory and coinhibitory signals. Thus, the rational manipulation of this pathway should aid in the development of safer and more effective drugs for a wide range of autoimmune-related inflammatory disorders. To this end, this review provides a summary on several recent patents associated with the LIGHT-HVEM-BTLA cosignaling system.

Unconventional ligand activation of herpesvirus entry mediator signals cell survival.

The herpesvirus entry mediator (HVEM; TNFRSF14) activates NF-kappaB 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-kappaB 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-kappaB. 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-kappaB 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.

The lymphotoxin-beta receptor is an upstream activator of NF-kappaB-mediated transcription in melanoma cells.

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

Evolutionarily divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway.

The herpesvirus entry mediator (HVEM), a member of the TNF receptor (TNFR) superfamily, can act as a molecular switch that modulates T cell activation by propagating positive signals from the TNF-related ligand LIGHT (TNFR superfamily 14), or inhibitory signals through the Ig superfamily member B and T lymphocyte attenuator (BTLA). Competitive binding analysis and mutagenesis reveals a unique BTLA binding site centered on a critical lysine residue in cysteine-rich domain 1 of HVEM. The BTLA binding site on HVEM overlaps with the binding site for the herpes simplex virus 1 envelope glycoprotein D, but is distinct from where LIGHT binds, yet glycoprotein D inhibits the binding of both ligands, potentially nullifying the pathway. The binding site on HVEM for BTLA is conserved in the orphan TNFR, UL144, present in human CMV. UL144 binds BTLA, but not LIGHT, and inhibits T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM. The demonstration that distinct herpesviruses target the HVEM-BTLA cosignaling pathway suggests the importance of this pathway in regulating T cell activation during host defenses.

Dimerizations of the wallaby gonadotropin-releasing hormone receptor and its splice variants.

Dimerization between wallaby GnRH-R and its splice variants was examined. A baculovirus-based fluorescence resonance energy transfer (Bv-FRET) assay was used to assess protein-protein interaction between wild type wallaby GnRH-R and splice variants (GnRH-RDelta1 and GnRH-RDelta2). FRET analysis demonstrated that GnRH-R, GnRH-RDelta1 or GnRH-RDelta2 are capable of assembling as homodimers. When GnRH-R is co-expressed with GnRH-RDelta1 or GnRH-RDelta2 splice variants, GnRH-R can form heterodimers with GnRH-RDelta1 and GnRH-RDelta2. GnRH agonist is not required for the initiation of dimerization. However, the addition of a GnRH agonist enhances the FRET signal in the GnRH-R homodimers, indicating that the GnRH agonist may be involved in modulating the extent of dimerization. In addition, this study reveals that dimerization of GnRH-R may be mediated by two or more protein interaction domains. One of them is probably located between amino acid residues 74 and 174, and the other one between residues 175 and 328. This is the first study to show dimerization between a wild type mammalian GnRH-R and its splice variants. It provides additional support for the potential involvement of splice variants in GnRH-R signaling.

Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein-protein interaction.

A new baculovirus-based fluorescence resonance energy transfer (Bv-FRET) assay for measuring multimerization of cell surface molecules in living cells is described. It has been demonstrated that gonadotropin-releasing hormone receptor (GnRH-R) was capable of forming oligomeric complexes in the plasma membrane under normal physiological conditions. The mouse gonadotropin-releasing hormone receptor GnRH-R was used to evaluate the efficiency and potential applications of this assay. Two chimeric constructs of GnRH-R were made, one with green fluorescent protein as a donor fluorophore and the other with enhanced yellow fluorescent protein as an acceptor fluorophore. These chimeric constructs were coexpressed in an insect cell line (BTI Tn5 B1-4) using recombinant baculoviruses. Energy transfer occurred from the excited donor to the acceptor when they were in close proximity. The association of GnRH-R was demonstrated through FRET and the fluorescence observed using a Leica TSC-SPII confocal microscope. FRET was enhanced by the addition of a GnRH agonist but not by an antagonist. The Bv-FRET assay constitutes a highly efficient, reliable and convenient method for measuring protein-protein interaction as the baculovirus expression system is superior to other transfection-based methods. Additionally, the same insect cell line can be used routinely for expressing any recombinant proteins of interest, allowing various combinations of molecules to be tested in a rapid fashion for protein-protein interactions. The assay is a valuable tool not only for the screening of new molecules that interact with known bait molecules, but also for confirming interactions between other known molecules.

Developmental expression and subcellular localization of wallaby gonadotropin-releasing hormone receptor and its splice variants.

The developmental expression of gonadotropin-releasing hormone receptor (GnRH-R) and its splice variants was examined in the gonads of tammar wallaby pouch young in order to elucidate the functional role of GnRH-R in the developing testis and ovary. Wallaby GnRH-R, like eutherian GnRH-Rs, contains three exons and two introns. In the present study, the transcripts of two splice variants (GnRH-R Delta 1 and GnRH-R Delta 2) were cloned from the pituitary. GnRH-R Delta 1 contained a 291 bp deletion from nucleotide positions 232 to 522 within exon 1. This transcript appears to be distinctive in the wallaby and has not been reported in other species. GnRH-R Delta 2 contained a 220 bp deletion from nucleotide positions 523 to 742, corresponding to exon 2. We examined the subcellular localization of the wild type GnRH-R and its splice variants with confocal microscopy, showing that both the wild type receptor and the splice variants were membrane-associated molecules. The different pattern of expression of the wild type receptor and the variants transcripts found in adult and neonatal tissues suggests a specific developmental regulation of the GnRH-R Delta 2 transcript. In addition, the developmental expression of the GnRH-R and GnRH-R Delta 1 transcripts showed a possible association with key physiological events during gonadal development in the wallaby pouch young, suggesting that GnRH-R may be involved in the regulation of early development in the testis and ovary.

Molecular cloning and tissue expression of the gonadotrophin-releasing hormone receptor in the tammar wallaby (Macropus eugenii).

Gonadotrophin-releasing hormone (GnRH) plays a pivotal role in the endocrine control of both reproduction and embryonic development. This first study of the marsupial GnRH receptor (GnRH-R) gene in the tammar wallaby provides information on the complex molecular events that regulate hypothalamic-pituitary-gonadal function in marsupials, and allows a comparison with eutherian mammals. Two identical wallaby GnRH-R cDNA clones were obtained, one isolated from cDNA generated from the testis of a 79-day-old pouch young and the other from the pituitary of an adult. Wallaby GnRH-R is composed of 328 amino acid residues. Sequence analysis showed that wallaby GnRH-R contains 7 transmembrane domains and is a member of the G protein-coupled receptor family. A putative protein kinase A phosphorylation site and a putative protein kinase C (PKC) phosphorylation site were found in the first intracellular loop, and an additional PKC phosphorylation site was located in the third intracellular loop. Comparisons with the eutherian GnRH-Rs show a greater diversity in the N-terminal extracellular domain. Wallaby GnRH-R has approximately 80% amino acid sequence homology with eutherian GnRH-Rs and 93% homology with the brush-tail possum, another member of the Diprotodontia semiorder.