Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-Stimulatory Signal Domain Exhibits Exhaustion-Resistant Properties.

Improving chimeric antigen receptor (CAR)-T cell therapeutic outcomes and expanding its applicability to solid tumors requires further refinement of CAR-T cells. We previously reported that CAR-T cells bearing a herpes virus entry mediator (HVEM)-derived co-stimulatory signal domain (CSSD) (HVEM-CAR-T cells) exhibit superior functions and characteristics. Here, we conducted comparative analyses to evaluate the impact of different CSSDs on CAR-T cell exhaustion. The results indicated that HVEM-CAR-T cells had significantly lower frequencies of exhausted cells and exhibited the highest proliferation rates upon antigenic stimulation. Furthermore, proliferation inhibition by programmed cell death ligand 1 was stronger in CAR-T cells bearing CD28-derived CSSD (CD28-CAR-T cells) whereas it was weaker in HVEM-CAR-T. Additionally, HVEM-CAR-T cells maintained a low exhaustion level even after antigen-dependent proliferation and exhibited potent killing activities, suggesting that HVEM-CAR-T cells might be less prone to early exhaustion. Analysis of CAR localization on the cell surface revealed that CAR formed clusters in CD28-CAR-T cells whereas uniformly distributed in HVEM-CAR-T cells. Analysis of CD3ζ phosphorylation indicated that CAR-dependent tonic signals were strongly sustained in CD28-CAR-T cells whereas they were significantly weaker in HVEM-CAR-T cells. Collectively, these results suggest that the HVEM-derived CSSD is useful for generating CAR-T cells with exhaustion-resistant properties, which could be effective against solid tumors.

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.

Lung-resident mesenchymal stem cells regulated the inflammatory responses in innate and adaptive immune cells through HVEM-BTLA pathway during ARDS.

Acute respiratory distress syndrome (ARDS) is an organ failure syndrome caused by overactivation of the immune system. Mesenchymal stem cells (MSCs) have been found to be effective in ARDS therapy due to their excellent immunomodulatory abilities; however, people are concerned about the safety of infusing exogenous cells. We found that rat lung-resident mesenchymal stem cells (LRMSCs) (Sca-1+CD45-CD31-) played important roles in regulating inflammation in the lungs during the pathogenesis of ARDS. LRMSCs could regulate the production of cytokines (TNF-α, MCP-1, and IL-10) by both innate and adaptive immune cells following LPS stimulation in vivo or in vitro. We also found that Herpes Virus Entry Mediator (HVEM) expression in LRMSCs enhanced the immunomodulatory ability of LRMSCs, and expression of the HVEM ligand B and T Lymphocyte Attenuator (BTLA) in innate and adaptive immune cells was required. The clarification of this immunoregulatory mechanism may provide evidence for ARDS therapy mediated by mobilizing endogenous MSCs in the future.

Fragments of gD Protein as Inhibitors of BTLA/HVEM Complex Formation-Design, Synthesis, and Cellular Studies.

One of the major current trends in cancer immunotherapy is the blockade of immune checkpoint proteins that negatively regulate the immune response. This has been achieved through antibodies blocking PD-1/PD-L1 and CTLA-4/CD80/CD86 interactions. Such antibodies have revolutionized oncological therapy and shown a new way to fight cancer. Additional (negative) immune checkpoints are also promising targets in cancer therapy and there is a demand for inhibitors for these molecules. Our studies are focused on BTLA/HVEM complex, which inhibits T-cell proliferation and cytokine production and therefore has great potential as a new target for cancer treatment. The goal of the presented studies was the design and synthesis of compounds able to block BTLA/HVEM interactions. For that purpose, the N-terminal fragment of glycoprotein D (gD), which interacts with HVEM, was used. Based on the crystal structure of the gD/HVEM complex and MM/GBSA analysis performed on it, several peptides were designed and synthesized as potential inhibitors of the BTLA/HVEM interaction. Affinity tests, ELISA tests, and cellular-based reporter assays were performed on these compounds to check their ability to bind to HVEM and to inhibit BTLA/HVEM complex formation. For leading peptides candidates, all-atom and subsequent docking simulations with a coarse-grained force field were performed to determine their binding modes. To further evaluate their potential as drug candidates, their stability in plasma and their cytotoxicity effects on PBMCs were assessed. Our data indicate that the peptide gD(1-36)(K10C-T29C) is the best candidate as a future drug. It interacts with HVEM protein, blocks the BTLA/HVEM interaction, and is nontoxic to cells. The present study provides a new perspective on the development of BTLA/HVEM inhibitors that disrupt protein interactions.

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.

Harringtonine Inhibits Herpes Simplex Virus Type 1 Infection by Reducing Herpes Virus Entry Mediator Expression.

Herpes simplex virus type 1 (HSV-1) infection induces various clinical disorders, such as herpes simplex encephalitis (HSE), herpes simplex keratitis (HSK), and genital herpes. In clinical intervention, acyclovir (ACV) is the major therapeutic drug used to suppress HSV-1; however, ACV-resistant strains have gradually increased. In the present study, harringtonine (HT) significantly inhibited infection of HSV-1 as well as two ACV-resistant strains, including HSV-1 blue and HSV-1 153. Time-of-drug addition assay further revealed that HT mainly reduced the early stage of HSV-1 infection. We also demonstrated that HT mainly affected herpes virus entry mediator (HVEM) expression as shown by qPCR, Western Blot, and Immunofluorescence. Collectively, HT showed antiviral activity against HSV-1 and ACV-resistant strains by targeting HVEM and could be a promising therapeutic candidate for mitigating HSV-1-induced-pathogenesis.

Clinical significance of herpes virus entry mediator expression in hepatitis B virus-related hepatocellular carcinoma.

Herpes virus entry mediator (HVEM) is overexpressed in several malignancies, including hepatocellular carcinoma (HCC). However, to the best of our knowledge, the clinical significance of HVEM in hepatitis B virus (HBV)-related HCC remains unclear. Thus, the present study aimed to explore the clinical significance of HVEM in HBV-related HCC. In the present study, HVEM expression was evaluated in HCC cell lines and HCC frozen samples. The prognostic value of HVEM was assessed in a cohort of 221 patients with HBV-related HCC, following radical resection. B- and T-lymphocyte attenuator (BTLA) expression in subsets of CD8+ T cells was determined via flow cytometry analysis. The results demonstrated high HVEM expression in HCC cell lines, and in HCC tissues compared with paired non-cancerous liver tissues. HVEM expression was demonstrated to be significantly associated with tumor encapsulation and vascular invasion. Furthermore, tumor HVEM status was significantly associated with infiltration of regulatory T cells, but not with CD8+ T cells. Notably, high HVEM expression in HCC was determined to be an independent predictor of an unfavorable outcome of patients with HCC following radical resection. Higher BTLA expression (the receptor of HVEM) was observed in both HCC-infiltrating CD8+ effector memory (CCR7- CD45RA-) and CD45RA+ effector memory (CCR7- CD45RA+) T cells in HCC tissues and blood compared with those in paired peritumor tissues or peripheral blood. Taken together, the results of the present study suggest that HVEM may serve a critical role in HBV-related HCC, most likely by promoting tumor progression and tumor immune evasion, thus the HVEM/BLTA signaling pathway may be a potential target in tumor immunotherapy.

High expression of herpes virus entry mediator is associated with poor prognosis in clear cell renal cell carcinoma.

Herpes virus entry mediator (HVEM), also called tumor necrosis factor receptor superfamily 14 (TNFRSF14), is highly expressed in various tumor tissues and plays critical roles in tumor biology. However, the role of HVEM in clear cell renal cell carcinoma (ccRCC) is unknown. This study evaluated the clinical importance of HVEM in patients with ccRCC. HVEM expression was assessed in fresh and 140 archived paraffin-embedded ccRCC tissue samples by quantitative RT-PCR, western blot, and immunohistochemical staining. HVEM expression was higher in ccRCC than in paired peritumor tissue. Kaplan-Meier analysis showed that high level of HVEM expression was associated with poor overall survival (OS) and disease-free survival (DFS) in patients with ccRCC (both P < 0.001). Multivariate analysis indicated that HVEM overexpression was independently prognostic of survival in ccRCC patients. Two novel nomogram systems were constructed by integrating HVEM expression and other clinical parameters to predict OS (c-index 0.75) and DFS (c-index 0.74) in these patients, with both having better predictive accuracy than traditional TNM (c-index 0.65 for OS and 0.639 for DFS) and Fuhrman (c-index 0.612 for OS and 0.641 for DFS) systems. In addition, HVEM silencing led to an observable reduction in tumor cells growth in vitro and in vivo. Taken together, these findings indicate that high HVEM expression is a novel and independent adverse predictor of clinical outcomes in patients with ccRCC and that HVEM may be a potential therapeutic target.

Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-stimulatory Signal Domain Exhibits High Functional Potency.

Chimeric antigen receptor (CAR) is a hybrid molecule consisting of an antigen-binding domain and a signal transduction domain. The artificial T cells expressing CAR (CAR-T cells) are expected to be a useful tool for treatment of various diseases, such as cancer. The addition of a co-stimulatory signal domain (CSSD) to CAR is shown to be critical for modulating CAR-T cell activities. However, the interplay among types of CSSDs, effector functions, and characteristics of CAR-T cells is largely unknown. To elucidate the interplay, we analyzed effector functions, differentiation to memory T cell subsets, exhaustion, and energy metabolism of the CAR-T cells with different CSSDs. Comparing to the CAR-T cells bearing a CD28- or 4-1BB-derived CSSD, which are currently used for CAR-T cell development, we found that the CAR-T cells with a herpes virus entry mediator (HVEM)-derived CSSD exhibited enhanced effector functions and efficient and balanced differentiation to both central and effector memory subsets, associated with an elevated energy metabolism and a reduced level of exhaustion. Thus, we developed the CAR-T cells bearing the CSSD derived from HVEM with high functional potency. The HVEM-derived CSSD may be useful for developing effective CAR-T cells.

BTLA/HVEM Signaling: Milestones in Research and Role in Chronic Hepatitis B Virus Infection.

B- and T-lymphocyte attenuator (BTLA) is an immune-regulatory receptor, similar to CTLA-4 and PD-1, and is mainly expressed on B-, T-, and all mature lymphocyte cells. Herpes virus entry mediator (HVEM)-BTLA plays a critical role in immune tolerance and immune responses which are areas of intense research. However, the mechanisms of the BTLA and the BTLA/HVEM signaling pathway in human diseases remain unclear. This review describes the research milestones of BTLA and HVEM in chronological order and their role in chronic HBV infection.

The immune checkpoint, HVEM may contribute to immune escape in non-small cell lung cancer lacking PD-L1 expression.

BACKGROUND: Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (B- and T-lymphocyte attenuator, BTLA) or activation of immune activity (LIGHT). The aim of this study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in NSCLC. METHODS: A TMA of 527 resected NSCLC samples and 56 NSCLC cell lines were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was optimized on the Dako Link48 autostainer using a polyclonal antibody from R&D Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 28-8 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used. RESULTS: HVEM expression in the NSCLC resected samples and cell lines revealed a positive H-score more than 1 was 18.6% (77/415) and 48.2% (27/56) respectively. HVEM expression was significantly higher in patients with lymph node N2 metastasis (25.5% vs 7.9% vs 17.5%, p = 0.046) when comparing with N1 or no lymph node metastasis, and was marginally significantly higher in patients with stage III/IV disease (24.5% vs 16.4%, p = 0.059). Subgroup analysis showed that HVEM (median 45 vs 36 months, p = 0.706) and PD-L1 expression (median 45 vs 48 months, p = 0.178) status was not predictive of overall survival. HVEM was found to have a significant negative correlation with PD-L1 expression (r=-0.274, p < 0.001) in patients with NSCLC and also a weak negative correlation in NSCLC cell lines (r=-0.162, p = 0.352). CONCLUSION: HVEM was found to be overexpressed in NSCLC patients of N2 lymph node metastasis or later stage and has a negative co-relationship with PD-L1 expression. HVEM was not prognostic for NSCLC patients.

Excess LIGHT contributes to placental impairment, increased secretion of vasoactive factors, hypertension, and proteinuria in preeclampsia.

Preeclampsia, a prevalent hypertensive disorder of pregnancy, is believed to be secondary to uteroplacental ischemia. Accumulating evidence indicates that hypoxia-independent mediators, including inflammatory cytokines and growth factors, are associated with preeclampsia, but it is unclear whether these signals directly contribute to placental damage and disease development in vivo. We report that LIGHT, a novel tumor necrosis factor superfamily member, is significantly elevated in the circulation and placentas of preeclamptic women compared with normotensive pregnant women. Injection of LIGHT into pregnant mice induced placental apoptosis, small fetuses, and key features of preeclampsia, hypertension and proteinuria. Mechanistically, using neutralizing antibodies specific for LIGHT receptors, we found that LIGHT receptors herpes virus entry mediator and lymphotoxin ß receptor are required for LIGHT-induced placental impairment, small fetuses, and preeclampsia features in pregnant mice. Accordingly, we further revealed that LIGHT functions through these 2 receptors to induce secretion of soluble fms-like tyrosine kinase-1 and endothelin-1, 2 well-accepted pathogenic factors in preeclampsia, and thereby plays an important role in hypertension and proteinuria in pregnant mice. Lastly, we extended our animal findings to human studies and demonstrated that activation of LIGHT receptors resulted in increased apoptosis and elevation of soluble fms-like tyrosine kinase-1 secretion in human placental villous explants. Overall, our human and mouse studies show that LIGHT signaling is a previously unrecognized pathway responsible for placental apoptosis, elevated secretion of vasoactive factors, and subsequent maternal features of preeclampsia, and reveal new therapeutic opportunities for the management of the disease.

Re-thinking the functions of IgA(+) plasma cells.

The intestinal mucosa harbors the largest population of antibody (Ab)-secreting plasma cells (PC) in the human body, producing daily several grams of immunoglobulin A (IgA). IgA has many functions, serving as a first-line barrier that protects the mucosal epithelium from pathogens, toxins and food antigens (Ag), shaping the intestinal microbiota, and regulating host-commensal homeostasis. Signals induced by commensal colonization are central for regulating IgA induction, maintenance, positioning and function and the number of IgA(+) PC is dramatically reduced in neonates and germ-free (GF) animals. Recent evidence demonstrates that the innate immune effector molecules tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) are required for IgA(+) PC homeostasis during the steady state and infection. Moreover, new functions ascribed to PC independent of Ab secretion continue to emerge, suggesting that PC, including IgA(+) PC, should be re-examined in the context of inflammation and infection. Here, we outline mechanisms of IgA(+) PC generation and survival, reviewing their functions in health and disease.

Clone and expression of human cytomegalovirus UL144 gene and its effects on dendritic cells / 第二军医大学学报

Objective: To construct a recombinant adenovirus vector carrying human cytomegalovirus (hCMV) UL144 gene and to explore the biological characteristics of UL144 gene-modified DCs. Methods: The UL144 gene was amplified from hCMV DNA, which was extracted from hCMV-DNA positive serum. The recombinant adenovirus vector carrying hCMV UL144 gene was constructed with AdEasy system and then transfected into HEK293 cells to create recombinant adenovirus Ad-UL144. The expression of inserted gene was identified by RT-PCR. The recombinant adenovirus was then transfected into mice myeloid dendritic cells. The surface proteins of dendritic cells were analyzed by FACS, and cytokines in supernatant were detected by ELISA. T cell proliferation stimulated by gene-modified DC was examined by 3H-TdR uptake assay. Results: The UL144 gene was successfully cloned into the pAdEasy-1 plasmid. The recombinant adenovirus Ad-UL144 was packed in HEK293 cells, with a viral titer of 3×10 10 pfu/ml. DCs infected with AdCMV-UL144 had markedly decreased surface expression of CD80, CD86 and I-Ad (P < 0.01). The contents of TNF-α, IL-6 and IL-1β were significantly decreased in the supernatant of AdCMV-UL144 modified DCs (P < 0.05). T cell proliferation ability induced by gene-modified DC was obviously lower than in the DC control group (P < 0.01). Conclusion: UL144-modified DCs can maintain a relative immature status, and have reduced stimulating activity upon the proliferation and activation of T cells in vitro.

Clone and expression of human cytomegalovirus UL144 gene and its effects on dendritic cells / 第二军医大学学报

Objective:To construct a recombinant adenovirus vector carrying human cytomegalovirus(hCMV) UL144 gene and to explore the biological characteristics of UL144 gene-modified DCs.Methods:The UL144 gene was amplified from hCMV DNA,which was extracted from hCMV-DNA positive serum.The recombinant adenovirus vector carrying hCMV UL144 gene was constructed with AdEasy system and then transfected into HEK293 cells to create recombinant adenovirus Ad-UL144.The expression of inserted gene was identified by RT-PCR.The recombinant adenovirus was then transfected into mice myeloid dendritic cells.The surface proteins of dendritic cells were analyzed by FACS,and cytokines in supernatant were detected by ELISA.T cell proliferation stimulated by gene-modified DC was examined by 3H-TdR uptake assay.Results:The UL144 gene was successfully cloned into the pAdEasy-1 plasmid.The recombinant adenovirus Ad-UL144 was packed in HEK293 cells,with a viral titer of 3?1010 pfu/ml.DCs infected with AdCMV-UL144 had markedly decreased surface expression of CD80,CD86 and I-Ad(P