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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.

Herpesvirus entry mediator (HVEM; TNFRSF14) is frequently mutated in human cancer with a high degree of mutations in follicular and diffuse large B cell lymphoma. In lymphocytes, HVEM receptors are activated by its ligands LIGHT (TNFSF14) and Lymphotoxin-α, and engage in bi-directional signaling with the receptors B and T lymphocyte attenuator (BTLA) and CD160. We hypothesized that mutations in HVEM disrupted binding to either of its ligands resulting in altered cell-intrinsic HVEM signaling in lymphoma, or in altered activation of BTLA or CD160 in tumor infiltrating lymphocytes. Here, we show that point mutations identified in human lymphoma were localized to the extracellular domain and specifically target ligand binding, resulting in preferential loss of HVEM interactions with CD160 and BTLA compared to LIGHT (TNFSF14). Disrupted binding correlated with loss of receptor functionality resulting in abrogated cell-intrinsic HVEM activation of NF-κB, as well as reduced CD160 activation of ERK and AKT phosphorylation by HVEM ligation. We next identified HVEM-activated CD160 and BTLA signaling pathways through AP/MS and phospho-proteomic analysis in NK and T cells. Activation of CD160 versus BTLA resulted in contrasting phosphorylation status in a number of phospho-proteins, illustrating opposing functions of these receptors. In a mouse model of lymphoma we tested the impact of BTLA versus CD160 in regulating antigen-specific anti-tumor responses, demonstrating regulatory effects of these receptors in vivo. Together these data illustrate how selective pressures within the tumor microenvironment may drive cancer mutations that alter HVEM network signaling in vivo, and indicate potential targets for onco-immunotherapy.

Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer’s Disease(1–4). Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.

Genetic studies have identified heritable linkages to psoriasis. However, complete identification of psoriasis susceptibility loci remains elusive. We have identified the HVEM-BTLA-CD160-LIGHT pathway as a critical regulator of innate and adaptive cell function in several autoimmune diseases, and recently in a mouse model of experimental psoriasis. Specifically, we have shown that the inhibitory receptor BTLA regulates the activity of innate γδ T cells, correlating with worsened dermatitis in BTLA-deficient animals, and attenuated disease in animals treated with agonistic BTLA antibodies. Additionally, our data indicates that the TNF receptor protein HVEM activates CD160 receptors in innate cells, promoting inflammatory signaling. More recently we have observed that skin inflammation is also worsened in animals lacking the TNF ligand for HVEM, LIGHT. In order to promote anti-inflammatory activities of HVEM, we sought to develop ligand specific biologics that could selectively engage inhibitory pathways through BTLA. Our initial studies showed that wild-type HVEM-Fc worsens disease. Additionally, we find that LIGHT-specific proteins that block HVEM-LIGHT interactions also worsen disease, consistent with our results in LIGHT-deficient animals, and identifying a unique regulatory role for LIGHT in skin inflammation. Finally, animals treated with proteins specific for BTLA and CD160 show reduced pathology compared to animals treated with wild-type HVEM-Fc. Through our efforts to synthesize ligand specific HVEM biologics, we have shown how select ablation of HVEM ligands can result in both pro- and anti-inflammatory signaling that can be targeted for the development of therapeutics for inflammatory diseases.