Formation Of High Endothelial Venules Research Articles

Abstract LB123: Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors

Abstract The presence of tertiary lymphoid structures (TLS) and associated high endothelial venules (HEV) in the tumor microenvironment strongly correlates with improved prognosis and treatment outcomes across a wide range of solid tumors. These clinical observations, now replicated repeatedly, highlight the therapeutic potential of induction of TLS and/or boosting TLS functions, which include acting as a point of entry and education for immune effector cells locally in the tumor. Lymphotoxin beta receptor (LTBR) signalling is essential for lymphoid structure development and the ectopic expression of its ligands, lymphotoxin αβ and LIGHT, is sufficient for TLS formation in vivo but challenging for therapeutic development. Here we present human bispecific antibodies (bsAbs) which conditionally agonize LTBR on co-engagement of fibroblast activation protein (FAP), a tumor microenvironment specific marker expressed by cancer associated fibroblasts. In vitro characterisation of these therapeutic bsAbs is presented, along with in vivo preclinical evaluation of surrogate bsAbs binding to mouse LTBR and mouse FAP in murine tumor models. Results - Therapeutic bsAbs were identified following an extensive screening campaign combining LTBR and FAP binding arms on an engineered human Fc with minimal FcyR binding. In vitro assays using primary cancer associated fibroblasts and LTBR positive cells demonstrated conditionally active therapeutic bsAbs potently activated LTBR in the presence of FAP expressing cells, whereas in the absence of FAP expressing cells no activity was observed. Surrogate bsAbs demonstrated monotherapy activity and led to tumor regressions in combination with anti-PDL1 therapy in an EMT6 mouse model, a syngeneic orthotopic model of breast cancer. Flow cytometry of endpoint tumors showed robust formation of HEVs and increased infiltration of T cells and B cells. In addition, we demonstrate that LTBR agonism, but not PD-L1 inhibition, leads to the formation of TLS structures containing organized lymphocyte aggregates with the appearance of germinal centres and accumulations of T and B cells in a mouse model of lung cancer. In conclusion, conditionally active FAP-LTBR bispecifics activate LTBR in the tumor microenvironment and induce HEV and TLS formation, leading to potent monotherapy activity in vivo, and to tumor regression in combination with PD-L1. These data support the development of FAP-LTBR bispecifics for the treatment of solid tumors as monotherapy and in combination with standard of care. Citation Format: Marta Lewandowska, Fevzi Demircioglu, Lucy Penfold, Rebecca B. Riddle, Sameer Sirohi, Floriane Laurent, Richard Brown, Sonia Bains, Dara Bevan, Emma Stanley, Jeanine Pignatelli, James W. Legg, Ray Jupp. Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB123.

Rapid Generation of hPSC-Derived High Endothelial Venule Organoids with In Vivo Ectopic Lymphoid Tissue Capabilities.

Bioengineering strategies for the fabrication of implantable lymphoid structures mimicking lymph nodes (LNs) and tertiary lymphoid structures (TLS) could amplify the adaptive immune response for therapeutic applications such as cancer immunotherapy. No method to date has resulted in the consistent formation of high endothelial venules (HEVs), which is the specialized vasculature responsible for naïve T cell recruitment and education in both LNs and TLS. Here orthogonal induced differentiation of human pluripotent stem cells carrying a regulatable ETV2 allele is used to rapidly and efficiently induce endothelial differentiation. Assembly of embryoid bodies combining primitive inducible endothelial cells and primary human LN fibroblastic reticular cells results in the formation of HEV-like structures that can aggregate into 3D organoids (HEVOs). Upon transplantation into immunodeficient mice, HEVOs successfully engraft and form lymphatic structures that recruit both antigen-presenting cells and adoptively-transferred lymphocytes, therefore displaying basic TLS capabilities. The results further show that functionally, HEVOs can organize an immune response and promote anti-tumor activity by adoptively-transferred T lymphocytes. Collectively, the experimental approaches represent an innovative and scalable proof-of-concept strategy for the fabrication of bioengineered TLS that can be deployed in vivo to enhance adaptive immune responses.

Effects of immunogenic cell death-inducing chemotherapeutics on the immune cell activation and tertiary lymphoid structure formation in melanoma.

The infiltration and activation of immune cells in the tumor microenvironment (TIME) affect the prognosis of patients with cancer. Tertiary lymphoid structure (TLS) formation favors tumour- infiltrating-lymphocyte (TIL) recruitment and is regarded as an important indicator of good prognosis associated with immunotherapy in patients with tumors. Chemotherapy is currently one of the most commonly used clinical treatment methods. However, there have been no clear report to explore the effects of different types of chemotherapy on TLS formation in the TIME. This study examined the effects of immunogenic cell death (ICD)-inducing chemotherapeutics on immune cells, high-endothelial venules (HEV), and TLSs in mouse melanomas. Doxorubicin (an ICD inducer), gemcitabine (non-ICD inducer), and a combination of the two drugs was delivered intra-peritoneally to B16F1-loaded C57BL/6 mice. The infiltration of immune cells into tumor tissues was evaluated using flow cytometry. HEV and TLS formation was assessed using immunohistochemistry and multiple fluorescent immunohistochemical staining. Doxorubicin alone, gemcitabine alone, and the two-drug combination all slowed tumor growth, with the combined treatment demonstrating a more pronounced effect. Compared with the control group, the doxorubicin group showed a higher infiltration of CD8+ T cells and tissue-resident memory T cells (TRM) and an increase in the secretion of interferon-γ, granzyme B, and perforin in CD8+ T subsets and activation of B cells and dendritic cells. Doxorubicin alone and in combination with gemcitabine decreased regulatory T cells in the TIME. Moreover, doxorubicin treatment promoted the formation of HEV and TLS. Doxorubicin treatment also upregulated the expression of programmed cell death protein (PD)-1 in CD8+ T cells and programmed cell death protein ligand (PD-L)1 in tumor cells. These results indicate that doxorubicin with an ICD reaction promotes TLS formation and increases PD-1/PD-L1 expression in tumor tissues. The results demonstrate the development of a therapeutic avenue using combined immune checkpoint therapy.

DC101, an anti-VEGFR2 agent, promotes high-endothelial venule formation and immune infiltration versus SAR131675 and fruquintinib

There is an increasing interest in combining immune checkpoint inhibitors (ICIs) with anti-angiogenic drugs to enhance their anti-tumor effects. In this study, three anti-angiogenic agents, DC101 (acting on VEGFR2), SAR131675 (acting on VEGFR3), and fruquintinib (a small-molecule inhibitor acting on multiple targets) were administered to B16F1-OVA-loaded C57BL/6 mice. Immune cells infiltration in the tumor tissues, vascular normalization, and high-endothelial venule (HEV) formation were assessed to provide evidence for drug combination. Both DC101 and fruquintinib significantly slowed the melanoma growth and increased the proportion of CD3+ and CD8+ T cells infiltration compared with SAR131675, of note, the effect of DC101 was more pronounced. Moreover, DC101 and fruquintinib increased the interferon-γ and perforin levels, meanwhile, DC101 increased the granzyme B levels, whereas fruquintinib and SAR131675 did not. Only the fruquintinib-treated group showed decreased regulatory T cells infiltration. We found upregulation of PD-L1 expression in tumor cells and CD45+ immune cells in DC101-treated group as well as upregulation of PD-1 expression on CD3+ T cells. However, fruquintinib only increased PD-L1 expression in tumors. Both DC101 and fruquintinib reduced the proportion of CD31+ vessels, while DC101 increased the ratio of α-SMA +/CD31+ cells and reduced the expression of HIF-1α more than fruquintinib. Moreover, DC101 enhanced the infiltration of dendritic cells and B cells, and local HEV formation. In conclusion, our data indicate that DC101 may be a better choice for the combined clinical application of ICIs and anti-angiogenic agents.

Lymphocyte access to lymphoma is impaired by high endothelial venule regression.

SummaryBlood endothelial cells display remarkable plasticity depending on the demands of a malignant microenvironment. While studies in solid tumors focus on their role in metabolic adaptations, formation of high endothelial venules (HEVs) in lymph nodes extends their role to the organization of immune cell interactions. As a response to lymphoma growth, blood vessel density increases; however, the fate of HEVs remains elusive. Here, we report that lymphoma causes severe HEV regression in mouse models that phenocopies aggressive human B cell lymphomas. HEV dedifferentiation occurrs as a consequence of a disrupted lymph-carrying conduit system. Mechanosensitive fibroblastic reticular cells then deregulate CCL21 migration paths, followed by deterioration of dendritic cell proximity to HEVs. Loss of this crosstalk deprives HEVs of lymphotoxin-β-receptor (LTβR) signaling, which is indispensable for their differentiation and lymphocyte transmigration. Collectively, this study reveals a remodeling cascade of the lymph node microenvironment that is detrimental for immune cell trafficking in lymphoma.

TLR7 Signaling Drives the Development of Sjögren's Syndrome.

Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease that affects predominately salivary and lacrimal glands. SS can occur alone or in combination with another autoimmune disease like systemic lupus erythematosus (SLE). Here we report that TLR7 signaling drives the development of SS since TLR8-deficient (TLR8ko) mice that develop lupus due to increased TLR7 signaling by dendritic cells, also develop an age-dependent secondary pathology similar to associated SS. The SS phenotype in TLR8ko mice is manifested by sialadenitis, increased anti-SSA and anti-SSB autoantibody production, immune complex deposition and increased cytokine production in salivary glands, as well as lung inflammation. Moreover, ectopic lymphoid structures characterized by B/T aggregates, formation of high endothelial venules and the presence of dendritic cells are formed in the salivary glands of TLR8ko mice. Interestingly, all these phenotypes are abrogated in double TLR7/8-deficient mice, suggesting that the SS phenotype in TLR8-deficient mice is TLR7-dependent. In addition, evaluation of TLR7 and inflammatory markers in the salivary glands of primary SS patients revealed significantly increased TLR7 expression levels compared to healthy individuals, that were positively correlated to TNF, LT-α, CXCL13 and CXCR5 expression. These findings establish an important role of TLR7 signaling for local and systemic SS disease manifestations, and inhibition of such will likely have therapeutic value.

The Role of Anti-angiogenesis in the Treatment Landscape of Non-small Cell Lung Cancer - New Combinational Approaches and Strategies of Neovessel Inhibition.

Tumor progression depends primarily on vascular supply, which is facilitated by angiogenic activity within the malignant tissue. Non-small cell lung cancer (NSCLC) is a highly vascularized tumor, and inhibition of angiogenesis was projected to be a promising therapeutic approach. Over a decade ago, the first anti-angiogenic agents were approved for advanced stage NSCLC patients, however, they only produced a marginal clinical benefit. Explanations why anti-angiogenic therapies only show modest effects include the highly adaptive tumor microenvironment (TME) as well as the less understood characteristics of the tumor vasculature. Today, advanced methods of in-depth characterization of the NSCLC TME by single cell RNA sequencing (scRNA-Seq) and preclinical observations enable a detailed characterization of individual cancer landscapes, allowing new aspects for a more individualized inhibition of angiogenesis to be identified. Furthermore, the tumor vasculature itself is composed of several cellular subtypes, which closely interact with other cellular components of the TME, and show distinct biological functions such as immune regulation, proliferation, and organization of the extracellular matrix. With these new insights, combinational approaches including chemotherapy, anti- angiogenic and immunotherapy can be developed to yield a more target-oriented anti-tumor treatment in NSCLC. Recently, anti-angiogenic agents were also shown to induce the formation of high endothelial venules (HEVs), which are essential for the formation of tertiary lymphoid structures, and key components in triggering anti-tumor immunity. In this review, we will summarize the current knowledge of tumor-angiogenesis and corresponding anti-angiogenic therapies, as well as new aspects concerning characterization of tumor-associated vessels and the resulting new strategies for anti-angiogenic therapies and vessel inhibition in NSCLC. We will further discuss why anti-angiogenic therapies form an interesting backbone strategy for combinational therapies and how anti-angiogenic approaches could be further developed in a more personalized tumor-oriented fashion with focus on NSCLC.

MyD88 signaling causes autoimmune sialadenitis through formation of high endothelial venules and upregulation of LT\u03b2 receptor-mediated signaling

Autoimmune sialadenitis (AS), chronic inflammation of the salivary glands (SGs) with focal lymphocyte infiltration, appears in autoimmune diseases such as Sjӧgren’s syndrome. The pathological role of MyD88-dependent innate immune signaling in autoimmune diseases including AS has been studied using mouse models, such as NOD mice. Although AS development in NOD mice was reported to be suppressed by Myd88 deficiency, its specific role remains unclear. Here, we determined the potent suppressive effects of Myd88 deficiency on AS development in lupus-prone B6/lpr mice, which have lymphoproliferation abnormalities, and also in NOD mice, which have no lymphoproliferation abnormalities. This indicates that MyD88 signaling triggers AS through both lymphoproliferation-dependent and -independent mechanisms. To address the MyD88-dependent lymphoproliferation-independent AS manifestation, SGs from C57BL/6 mice were analyzed. Remarkable upregulation of Glycam1 and high endothelial venule (HEV)-associated changes were unexpectedly found in Myd88+/+ mice, compared with Myd88−/− mice. MyD88-dependent HEV-associated changes were also observed in NOD mice. Additionally, Lta, Ltb, and Ltbr in SGs of NOD mice were lowered by Myd88 deficiency. Interestingly, LTβR-induced HEV-associated gene expression in cultured cells was impaired by Myd88 deficiency. Our findings highlight novel roles for MyD88 in AS development, which imply the existence of MyD88-dependent HEV formation in ectopic lymphoid neogenesis.

Combined antiangiogenic and anti-PD-L1 therapy stimulates tumor immunity through HEV formation.

Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ-expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti-PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin β receptor (LTβR) signaling. Further activation of LTβR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTβR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti-PD-L1 therapy. Together, our preclinical studies provide evidence that anti-PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti-PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction.

Role of vascular endothelial growth factor, survivin, and inducible nitric oxide synthase expression in psoriasis: an immunohistochemical study

Background Psoriasis (PS) is a common chronic, relapsing, immune-mediated disease involving the skin and joints of genetically predisposing individuals. Despite numerous studies, the pathogenesis of PS has not been fully elucidated. However, many pathogenic theories have been suggested. There is much evidence that PS is a polygenic disease modified to expression by triggering factors. PS is characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes, lymphocyte infiltration consisting mostly of T lymphocytes, and various endothelial vascular changes in the dermal layer, such as angiogenesis, dilation, and high endothelial venule formation. Vascular endothelial growth factor (VEGF) is a major regulator of physiological and pathological angiogenesis, causing aberrant angiogenesis and vascular leakage in the upper dermis; it may also contribute to keratinocyte proliferation and epidermal barrier homeostasis in PS. Inducible nitric oxide synthase (iNOS) was found to be expressed in the keratinocytes of psoriatic lesions; in addition, there is increased expression of iNOS-specific mRNA transcripts. Survivin upregulation in psoriatic lesion in comparison with normal skin was evident, suggesting its role in the pathogenesis of PS. The aim of this study was to investigate the immunohistochemical expression of VEGF, survivin, and iNOS in psoriatic skin and to detect their role in the pathogenesis of PS. Patients and methods This study was carried out on 20 patients with PS vulgaris. Immunohistochemical reactions were carried out using the streptavidin-biotin immunoperoxidase system in this study to detect the extent of expression of VEGF, survivin, and iNOS in each specimen of the skin biopsy. Results A strong VEGF expression through the epidermis (mean 46.4 ΁ 19.7) was observed. VEGF was significantly upregulated in psoriatic skin in comparison with normal healthy skin ( P P P Conclusion VEGF, survivin, and iNOS appeared to be important factors in the pathogenesis of PS.

Endothelial cell–specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)-restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC-lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice.

Salivary glands in patients with Sjögren's syndrome (SS) develop ectopic lymphoid structures (ELS) characterized by B/T cell compartmentalization, the formation of high endothelial venules, follicular dendritic cell networks, functional B cell activation with expression of activation-induced cytidine deaminase, as well as local differentiation of autoreactive plasma cells. The mechanisms that trigger ELS formation, autoimmunity, and exocrine dysfunction in SS are largely unknown. In this article, we present a novel model of inducible ectopic lymphoid tissue formation, breach of humoral self-tolerance, and salivary hypofunction after delivery of a replication-deficient adenovirus-5 in submandibular glands of C57BL/6 mice through retrograde excretory duct cannulation. In this model, inflammation rapidly and consistently evolves from diffuse infiltration toward the development of SS-like periductal lymphoid aggregates within 2 wk from AdV delivery. These infiltrates progressively acquire ELS features and support functional GL7(+)/activation-induced cytidine deaminase(+) germinal centers. Formation of ELS is preceded by ectopic expression of lymphoid chemokines CXCL13, CCL19, and lymphotoxin-β, and is associated with development of anti-nuclear Abs in up to 75% of mice. Finally, reduction in salivary flow was observed over 3 wk post-AdV infection, consistent with exocrine gland dysfunction as a consequence of the inflammatory response. This novel model has the potential to unravel the cellular and molecular mechanisms that regulate ELS formation and their role in exocrine dysfunction and autoimmunity in SS.

Antigen‐dependent rescue of nose‐associated lymphoid tissue (NALT) development independent of LTβR and CXCR5 signaling

Nose-associated lymphoid tissue (NALT) in the rodent upper respiratory tract develops postnatally and is considered to be independent of several factors known to be involved in the organogenesis of LN and Peyer's patches (PP). In this study we demonstrate that at least two different pathways result in NALT development. Following NALT anlage formation the intrinsic pathway relies on a signaling cascade including those mediated through the chemokine receptor CXCR5 and the lymphotoxin beta receptor (LTbetaR). This allows for the formation of high endothelial venules and thereby the recruitment of lymphocytes into NALT. Alternatively, high endothelial venule formation and lymphocyte recruitment can be induced in the NALT anlage by environmental signals, which are independent of LT-betaR and chemokine receptor CXCR5 signaling but in part rely on CD40 ligand. Thus, our study identifies a novel mechanism that facilitates the rescue of NALT development at late stages in adult life independent of the canonical LTbetaR-CXCR5 signaling axis.

Mycobacterium tuberculosisTriggers Formation of Lymphoid Structure in Murine Lungs

The hallmark of pulmonary tuberculosis is the granuloma, which consists predominantly of lymphocytes and macrophages and promotes immune-cell interaction with the causative pathogen, Mycobacterium tuberculosis. Granuloma formation is a highly organized process, which depends on leukocyte recruitment facilitated by adhesion molecules and chemokines. Thus, during chronic experimental tuberculosis, granulomata display characteristics of lymphoid structures comprising follicular aggregation of B cells, formation of high endothelial venules, presence of follicular dendritic cells, and expression of the homeostatic chemokines CXCL13 and CCL19. CCR7-/- mice, which are deficient in CCL19 and CCL21 signaling, exhibit increased local inflammatory infiltrates but no follicular B-cell aggregation within those lymphoid structures. However, CCR7-deficient mice are fully capable to control pulmonary tuberculosis; at time points later than 6 weeks postinfection, they carry a lower bacterial load in peripheral organs. Our results show that, during chronic pulmonary tuberculosis in mice, the homeostatic chemokine signaling-network contributes to spatial organization of the granulomatous response, which participates in both containment of M. tuberculosis and the latter's dissemination to other organs.

The role of the endothelium in rheumatoid arthritis (RA)

RA is a systemic disease characterised by inflammation and destruction of synovial joints. The synovial microvasculature undergoes two distinct morphological changes - namely the formation of high endothelial venules (HEVs) and the development of new blood vessels (angiogenesis or neovascularisation) which contribute to joint inflammation and destruction. HEVs are specialised post-capillary venules lined by cuboidal endothelium, normally restricted in distribution to lymphoid tissues. In RA HEVs are found in proximity to extravascular collections of lymphocytes, and may facilitate lymphocyte entry to the inflamed synovium. The targeting of lymphocytes to rheumatoid synovium is thought to be due to the interaction of lymphocyte homing receptors with unique acceptor molecules expressed on synovial HEVs. Rheumatoid pannus is comprised of a fibrovascular proliferative granulation tissue that progressively invades articular cartilage. Normally hyaline articular cartilage is avascular and resistant to neovascularisation. In RA the synovium overcomes the anti-angiogenic effects of articular cartilage. Although the cause of neovascularisation is unknown angiogenesis promoters including macrophages and mast cells, as well as pro-angiogenic cytokines such as tumour necrosis factor a and transforming growth factor β are present within the rheumatoid joint and could participate in this process. Furthermore a loss of anti-angiogenic activity of rheumatoid articular cartilage may contribute to neovascularisation. In summary, microvascular changes in the rheumatoid synovium lead to the facilitation of lymphocyte trafficking and to neovascularisation with inflammatory and destructive sequelae. A better understanding of their control may lead to novel approaches to therapy.

Cadmium effects on the female reproductive tract∗

The effects of Cd exposure on ovaries and placenta are reviewed. The effects of Cd exposure on the vasculature of the uterus are summarised and the results of the latest experiments (exposure to 5 and 15 ppm in drinking water) are described. The results are compared with electronmicroscopie data of s.c. exposure during 80 weeks. It was concluded that Cd interacts with the vessels, leading to an inflammatory response, that finally causes the formation of High Endothelial Venules (HEV). The reactions are dose dependent, but due to a continuous renewal of endothelium the effects seem to be more or less time independent.