Studies with a candidate vaccine deleted in glycoprotein D (ΔgD-2) for herpes simplex virus (HSV) prevention uncovered a role for herpes virus entry mediator (HVEM) in mediating antibody-dependent cell-mediated killing (ADCK) of virally infected cells. Antibodies elicited by ΔgD-2 passively protect WT but not Fc γ receptor (FcγR) or HVEM knockout (KO) mice. The goals of this study were to identify which cells mediate ADCK and the role of HVEM signaling. Using HVEM ligand and conditional cell-type-specific HVEM-KO mice combined with in vitro mouse and human cytolytic assays, we demonstrate that ADCK of HSV-infected cells is mediated primarily by neutrophils and requires their expression of HVEM and its ligand, LIGHT. Cytolysis is not associated with granzyme and perforin production but occurs by a trogocytosis-like pathway. Pharmacological inhibition of myosin light-chain kinase (MLCK), which mediates trogocytosis, inhibits cytolysis. Similar results were obtained when human neutrophils were cocultured with HSV-infected cells opsonized with ADCK-containing human immune serum or with breast cancer cells treated with an anti-HER2 trogocytosis mediating antibody. Killing was significantly reduced when an MLCK inhibitor or blocking antibodies to CD16a, HVEM, or LIGHT were added. Together, these results define a mechanism of HVEM-enhanced FcγR-mediated neutrophil-dependent ADCK of targets cells.

The herpesvirus entry mediator (HVEM) (TNFRSF14) engagement of the checkpoint inhibitory receptor B and T lymphocyte attenuator (BTLA) limits immune responses of T and B lymphocytes. HVEM and BTLA form signaling complexes in trans and when coexpressed, complexes in cis, creating a unique immune checkpoint. The function of the HVEM-BTLA cis-complex is not well understood primarily due to a lack of reagents that specifically measure the HVEM-BTLA cis-complex. We describe here a method to generate antibodies to receptor-ligand complexes using fusion immunogens, in this case, a BTLA-HVEM fusion protein. We identified 2 closely related antibodies that specifically recognize the HVEM-BTLA complex on the cell surface. In experiments utilizing the anti-HVEM-BTLA complex-specific antibody together with subunit-specific BTLA monoclonal antibodies, we were able to determine the precise ratio of free to cis-complexed BTLA on subpopulations of human lymphocytes. This is the first direct quantification of the HVEM-BTLA cis-complex. The method described here should apply to the detection of other receptor-ligand complexes.

B cell-rich tertiary lymphoid structures (TLS) are associated with favorable prognosis and positive response to immunotherapy in cancer. Here we show that simultaneous activation of innate immune effectors, STING and lymphotoxin-β receptor (LTβR), results in CD8+ T cell-dependent tumor suppression while inducing high endothelial venule development and germinal center-like B cell responses in tumors to generate functional TLS in a T cell-dependent manner. In a neoadjuvant setting, activation of STING and LTβR by their agonists effectively immunized mice against tumor recurrence, leading to long-term survival. STING activation alone was insufficient for inducing B cell-containing TLS or eliciting long-term therapeutic effects. However, when combined with LTβR activation, it improved the fitness of TLS with B cell expansion and maturation to IgG-producing long-lived plasma cells and memory cells, increased CD4+ T cell recruitment and memory CD8+ T cell expansion, and shifted the TH2/TH17 balance, resulting in the potentiation of humoral and cellular immunity against tumors. These findings suggest a therapeutic approach of simultaneously activating STING and lymphotoxin pathways. Sawada et al. show simultaneous activation of the STING and lymphotoxin beta receptor signaling induces B cell-activating germinal center responses within tumor environment and enhances antitumor responses.

Abstract B cells are emerging as key players in anti-tumor immunity. Their ability to produce antibodies against tumor-associated antigens makes them potent agents in cancer immunotherapy. However, B cell-based therapies remain underutilized compared with T cell-focused approaches. Tumor-infiltrating B cells can form tertiary lymphoid structures (TLS) that resemble lymphoid follicles of inflamed lymph nodes. Significant evidence indicates that TLS are associated with enhanced anti-tumor immunity, and the presence of TLS correlates with improved prognosis and better therapeutic responses across various cancers. However, the development of B cell-dependent anti-tumor immunity might be generated mainly in the tumor-draining lymph nodes, and TLS formation may be a bystander effect of the strong local immune responses. Thus, direct evidence for TLS-B cells contributing to anti-tumor immunity is lacking. Recently, our laboratory discovered that simultaneous activation of innate immune effectors, stimulator of interferon genes (STING) and lymphotoxin beta receptor (LTβR), by their agonists induces mature TLS with germinal center B cell responses in various mouse tumor models. In the current study, we investigated the role of TLS using CD79a knockout mice, which cannot develop TLS due to B cell deficiency. We also leveraged hybridoma technology to generate monoclonal antibodies from TLS-B cells for potential tumor targeting. We show that, in wild type mice, the STING activation improved the fitness of LTβR-induced TLS with B cell maturation to IgG+ long-lived plasma cells and memory cells. This treatment reduced the tumor size and heightened the efficacy of anti-PD-1 immune checkpoint inhibition, leading to complete cure. When used as neoadjuvant therapy, it prevented tumor recurrence and metastasis with 100% survival post-tumor rechallenge, indicating the effective immunization against tumors. Functional TLS formed in different tumor types and anatomical sites using this approach. Hybridoma clones developed from TLS-B cells of these tumors generated monoclonal IgG antibodies targeting mouse pancreatic cancer cells, which effectively induced NK cell-mediated cytotoxicity. In contrast, B cell-deficient CD79a KO mice failed to develop TLS while still being capable of developing high endothelial venules in primary tumors. T cell infiltration did not appear to be reduced by the B cell deficiency. However, these mice showed significantly reduced survival (40%) after tumor rechallenge, underscoring the importance of TLS-B cells for coordinated anti-tumor immune responses. These findings underscore the critical role of B cells in the development of functional TLS and strong anti-tumor immunity. The STING and LTβR agonist combination therapeutically induces TLS to produce tumor-specific high-affinity antibodies with significant tumor-killing potential. This approach highlights a promising strategy for targeting immune-cold tumors that are refractory to standard immunotherapies, paving the way for innovative immunotherapies that harness the power of B cells. Citation Format: Maxwell Duah, Yasuhiro Kikuchi, Tomoko Stansel, Krisztian Csomos, Nobuyoshi Hiraoka, Jolan Walter, Carl F Ware, Masanobu Komatsu. Intratumoral B cells are essential for anti-tumor immune responses of tertiary lymphoid structures: Impact of STING and LTβR activation in pancreatic cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR001.

The first anti-tumour necrosis factor (TNF) monoclonal antibody, infliximab (Remicade), celebrated its 25th anniversary of FDA approval in 2023. Inhibitors of TNF have since proved clinically efficacious at reducing inflammation associated with several autoimmune diseases, including rheumatoid arthritis, psoriasis and Crohn's disease. The success of TNF inhibitors raised unrealistic expectations for targeting other members of the TNF superfamily (TNFSF) of ligands and their receptors, with difficulties in part related to their more limited, variable expression and potential redundancy. However, there has been a resurgence of interest and investment, with many of these cytokines or their cognate receptors now under clinical investigation as targets for modulation of autoimmune and inflammatory diseases, as well as cancer. This Review assesses TNFSF-targeted biologics currently in clinical development for immune system-related diseases, highlighting ongoing challenges and future directions.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a cancer with low survival rates and currently no immunotherapy options. However, many tumors from PDAC patients can be heavily infiltrated with T and B cells associated with favorable survival outcomes indicating anti-tumor immunity is functional in certain patients. In some of these lymphocyte-inflamed PDAC tumors, spontaneous organization of tertiary lymphoid structures (TLS) is evident upon surgical resection and these patients have a clear survival advantage. TLS are predictive of immune checkpoint blockade response in some cancer types, yet PDAC patients, with or without TLS, are still insensitive to these strategies. To address this paradox, we evaluated surgically resected PDAC tumors from previously untreated patients and discovered that tumors with TLS (TLS+) recruited T and B cells with specific anti-tumor and memory phenotypes suggesting immunosurveillance of disease was enhanced. Furthermore, a subset of TLS+ patients contained germinal centers and improved humoral immune function corresponding to an increased neoantigen burden and long-term survival. When assessing gene expression differences between TLS+ and TLS- patients, operable inflammatory and immunosuppressive pathways emerged that could be therapeutically exploited to activate TLS neogenesis. To address these mechanistic questions, we have developed a method for inducing TLS formation in implantable mouse PDAC tumors in the orthotopic setting utilizing an antibody that agonizes the lymphotoxin beta receptor (LTBR) in mice bearing established PDAC tumors. These TLS are complexed with T cells, B cells, CXCL13+ cells, and PNAd+ HEV associated with reduced tumor growth, presence of lymphocyte-recruiting cancer associated fibroblasts and increases in anti-tumor T and B cell phenotypes. Interestingly, some mouse PDAC cell lines are resistant to TLS formation by LTBR agonism while others are susceptible offering a model to study patient heterogeneity. We will use this TLS+ PDAC mouse model to address how TLS may directly improve antigen-specific T cell and B cell immunity and elucidate the pathways that regulate TLS formation. These data will offer new strategies to overcome immunotherapy resistance in cancer patients. Citation Format: Shrijan Khanal, Olivia Harder, Elijah Kirschstein, Morgan Mack, Carl Ware, Michael Gough, Kristina Young, Andrew J. Gunderson. Tertiary lymphoid structures in pancreatic cancer: Biomarkers of immunogenic patients or direct contributors to anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7464.

Impact of Treg depletion on LN HEV remodelling

LSFM imaging of Treg depleted and T-cell depleted fibrosarcomas.

Global overview of intra-tumoral HEVs in a 4T1 regressor tumour following PI-3065 treatment.

2D assessment of fibrosarcoma establishment on LN HEVs

Global overview of intra-tumoral HEVs in a 4T1 regressor tumour following PI-3065 treatment.

Impact of Treg depletion and LTβR agonist treatment on the immune cell organisation in fibrosarcoma tumours.

LSFM imaging of Treg depleted fibrosarcomas.

S1. Tumor HEV identified by PNAd staining are also positive for MAdCAM-1. S2. Efficacy of monoclonal antibody treatments to deplete immune cell subsets. S3. Splenic Marginal Zone B cells are profoundly decreased after treatment with LTbetaR.Fc. S4. Splenic Follicular Dendritic Cells and MAdCAM-1 staining are lost after treatment with LTbetaR.Fc or TNFRII.Ig. S5. Lymph Node architecture is disrupted following treatment with LTbetaR.Fc or TNFRII.Ig. S6. Tumor HEV identified by PNAd staining following blockade of LTbetaR or TNFR signalling are also positive for MAdCAM-1. S7. Agonism of LTβR induces formation of High Endothelial Venules in Treg replete tumors, but without concomitant increased T cell infiltration and reduced tumor growth. S8. Relative gene expression of TNF and LTalpha by intratumoral CD4+ and CD8+ T cells and dendritic cells (DC). Data are expressed as fold change in gene expression relative to splenic B cells, and represent two independent experiments.

Global overview of intra-tumoral HEVs in a 4T1 non-regressor tumour following PI-3065 treatment.

Global overview of intra-tumoral HEVs in 4T1 tumours following PI-3065 treatment.

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 medi-ator (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 pro-vide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.

The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes contribute to immunoregulation. Here, we show that Btla inhibits activation of genes regulating metabolism and cytokine signaling, including Il6 and Hif1a, indicating a regulatory role in humoral immunity. Within mucosal Peyer's patches, we find T-cell-expressed Btla-regulated Tfh cells, while Btla in T or B cells regulates GC B cell numbers. Treg-expressed Btla is required for cell-intrinsic Treg homeostasis that subsequently controls GC B cells. Loss of Btla in lymphocytes results in increased IgA bound to intestinal bacteria, correlating with altered microbial homeostasis and elevations in commensal and pathogenic bacteria. Together our studies provide important insights into how Btla functions as a checkpoint in diverse conventional and regulatory lymphocyte subsets to influence systemic immune responses.

Many coinhibitory receptors are absent from naïve T cells and upregulated upon activation. In contrast, there are a small number of coinhibitory receptors expressed constitutively by naïve T cells, including CD5, BTLA, and VISTA. The relationship between these constitutively expressed coinhibitors is unknown. We examined the relationship between the constitutively expressed BTLA and CD5 in T cell ontogeny. We found an inverse relationship between CD5 and BTLA expression levels, with low BTLA expression in the thymus and higher BTLA expression in the periphery, corresponding with high and low CD5 expression, respectively. To determine if there is a causal relationship between BTLA expression and CD5 expression, we examined CD5 expression in wild type (WT) vs. btla−/− T cells. Interestingly, btla−/− T cells consistently expressed higher levels of CD5 both in thymic and splenic CD4 and CD8 T cells, indicating that BTLA expression directly or indirectly determines the level of CD5 expression broadly across T cells. In contrast, the loss of an inducible coinhibitor, PD-1, only affected CD5 levels on splenic CD8+ T cells. The loss of BTLA expression early in ontogeny might alter the TCR repertoire indirectly affecting CD5 levels, or instead its loss might affect CD5 expression rapidly within mature T cells. To examine the latter, we deleted btla by administering tamoxifen to adult btlafl/fl CreERT2+/− mice and WT CreERT2+/− controls. Loss of btla in adults led only to a transiently heightened CD5 expression. Together our data indicates that loss of BTLA early, but not later in ontogeny, leads to a long-term increase in CD5 expression by T cells. Such calibration of CD5 levels early in ontogeny might serve to reduce autoimmunity. Funded by the CIHR, NSERC, and an AAI fellowship; btlafl/fl mice provided by the La Jolla Institute for Immunology.

Advances in understanding the physiologic functions of the tumor necrosis factor superfamily (TNFSF) of ligands, receptors, and signaling networks are providing deeper insight into pathogenesis of infectious and autoimmune diseases and cancer. LIGHT (TNFSF14) has emerged as an important modulator of critical innate and adaptive immune responses. LIGHT and its signaling receptors, herpesvirus entry mediator (TNFRSF14), and lymphotoxin β receptor, form an immune regulatory network with two co-receptors of herpesvirus entry mediator, checkpoint inhibitor B and T lymphocyte attenuator, and CD160. Deciphering the fundamental features of this network reveals new understanding to guide therapeutic development. Accumulating evidence from infectious diseases points to the dysregulation of the LIGHT network as a disease-driving mechanism in autoimmune and inflammatory reactions in barrier organs, including coronavirus disease 2019 pneumonia and inflammatory bowel diseases. Recent clinical results warrant further investigation of the LIGHT regulatory network and application of target-modifying therapeutics for disease intervention.