VISTA Targeting of T-cell Quiescence and Myeloid Suppression Overcomes Adaptive Resistance.

V domain immunoglobulin suppressor of T-cell activation (VISTA) is a premier target for cancer treatment due to its broad expression in many cancer types and enhanced expression upon development of adaptive immune checkpoint resistance. In the CT26 colorectal cancer model, monotherapy of small tumors with anti-VISTA resulted in slowed tumor growth. In a combination therapy setting, large CT26 tumors showed complete adaptive resistance to anti-PD-1/CTLA-4, but inclusion of anti-VISTA led to rejection of half the tumors. Mechanisms of enhanced antitumor immunity were investigated using single-cell RNA sequencing (scRNA-seq), multiplex image analysis, and flow cytometry of the tumor immune infiltrate. In both treatment models, anti-VISTA upregulated stimulated antigen presentation pathways and reduced myeloid-mediated suppression. Imaging revealed an anti-VISTA stimulated increase in contacts between T cells and myeloid cells, further supporting the notion of increased antigen presentation. scRNA-seq of tumor-specific CD8+ T cells revealed that anti-VISTA therapy induced T-cell pathways highly distinct from and complementary to those induced by anti-PD-1 therapy. Whereas anti-CTLA-4/PD-1 expanded progenitor exhausted CD8+ T-cell subsets, anti-VISTA promoted costimulatory genes and reduced regulators of T-cell quiescence. Notably, this is the first report of a checkpoint regulator impacting CD8+ T-cell quiescence, and the first indication that quiescence may be a target in the context of T-cell exhaustion and in cancer. This study builds a foundation for all future studies on the role of anti-VISTA in the development of antitumor immunity and provides important mechanistic insights that strongly support use of anti-VISTA to overcome the adaptive resistance seen in contemporary treatments involving PD-1 and/or CTLA-4. See related Spotlight by Wei, p. 3.

VISTA Re-programs Macrophage Biology Through the Combined Regulation of Tolerance and Anti-inflammatory Pathways.

We present the novel finding that V-domain Ig suppressor of T cell activation (VISTA) negatively regulates innate inflammation through the transcriptional and epigenetic re-programming of macrophages. Representative of VISTA re-programming is the ability of VISTA agonistic antibodies to augment LPS tolerance and reduce septic shock lethality in mice. This anti-inflammatory effect of anti-VISTA was mimicked in vitro demonstrating that anti-VISTA treatment caused a significant reduction in LPS-induced IL-12p40, IL-6, CXCL2, and TNF; all hallmark pro-inflammatory mediators of endotoxin shock. Even under conditions that typically "break" LPS tolerance, VISTA agonists sustained a macrophage anti-inflammatory profile. Analysis of the proteomic and transcriptional changes imposed by anti-VISTA show that macrophage re-programming was mediated by a composite profile of mediators involved in both macrophage tolerance induction (IRG1, miR221, A20, IL-10) as well as transcription factors central to driving an anti-inflammatory profile (e.g., IRF5, IRF8, NFKB1). These findings underscore a novel and new activity of VISTA as a negative checkpoint regulator that induces both tolerance and anti-inflammatory programs in macrophages and controls the magnitude of innate inflammation in vivo.

Endocytosis of very low-density lipoproteins: an unexpected mechanism for lipid acquisition by breast cancer cells.

We previously described the expression of CD36 and LPL by breast cancer (BC) cells and tissues and the growth-promoting effect of VLDL observed only in the presence of LPL. We now report a model in which LPL is bound to a heparan sulfate proteoglycan motif on the BC cell surface and acts in concert with the VLDL receptor to internalize VLDLs via receptor-mediated endocytosis. We also demonstrate that gene-expression programs for lipid synthesis versus uptake respond robustly to triglyceride-rich lipoprotein availability. The literature emphasizes de novo FA synthesis and exogenous free FA uptake using CD36 as paramount mechanisms for lipid acquisition by cancer cells. We find that the uptake of intact lipoproteins is also an important mechanism for lipid acquisition and that the relative reliance on lipid synthesis versus uptake varies among BC cell lines and in response to VLDL availability. This metabolic plasticity has important implications for the development of therapies aimed at the lipid dependence of many types of cancer, in that the inhibition of FA synthesis may elicit compensatory upregulation of lipid uptake. Moreover, the mechanism that we have elucidated provides a direct connection between dietary fat and tumor biology.-.

Defining the Signature of VISTA on Myeloid Cell Chemokine Responsiveness.

The role of negative checkpoint regulators (NCRs) in human health and disease cannot be overstated. V-domain Ig-containing Suppressor of T-cell Activation (VISTA) is an Ig superfamily protein predominantly expressed within the hematopoietic compartment and has been studied for its role in the negative regulation of T cell responses. The findings presented in this study show that, unlike all other NCRs, VISTA deficiency dramatically impacts on macrophage cytokine and chemokine production, as well as the chemotactic response of VISTA-deficient macrophages. A select group of inflammatory chemokines, including CCL2, CCL3, CCL4, and CCL5, was strikingly elevated in culture supernatants from VISTA KO macrophages. VISTA deficiency also altered chemokine receptor recycling and profoundly disrupted myeloid chemotaxis. The impact of VISTA deficiency on chemotaxis in vivo was apparent with the reduced ability of both KO macrophages and MDSCs to migrate to the tumor microenvironment. This is the first demonstration of an NCR impacting on myeloid mediator production and chemotaxis, and will guide the use of anti-VISTA therapeutics to manipulate the chemotaxis of inflammatory macrophages or immunosuppressive MDSCs in inflammatory diseases and cancer.

HS3ST1 genotype regulates antithrombin's inflammomodulatory tone and associates with atherosclerosis.

The HS3ST1 gene controls endothelial cell production of HSAT+ - a form of heparan sulfate containing a specific pentasaccharide motif that binds the anticoagulant protein antithrombin (AT). HSAT+ has long been thought to act as an endogenous anticoagulant; however, coagulation was normal in Hs3st1-/- mice that have greatly reduced HSAT+ (HajMohammadi et al., 2003). This finding indicates that HSAT+ is not essential for AT's anticoagulant activity. To determine if HSAT+ is involved in AT's poorly understood inflammomodulatory activities, Hs3st1-/- and Hs3st1+/+ mice were subjected to a model of acute septic shock. Compared with Hs3st1+/+ mice, Hs3st1-/- mice were more susceptible to LPS-induced death due to an increased sensitivity to TNF. For Hs3st1+/+ mice, AT treatment reduced LPS-lethality, reduced leukocyte firm adhesion to endothelial cells, and dilated isolated coronary arterioles. Conversely, for Hs3st1-/- mice, AT induced the opposite effects. Thus, in the context of acute inflammation, HSAT+ selectively mediates AT's anti-inflammatory activity; in the absence of HSAT+, AT's pro-inflammatory effects predominate. To explore if the anti-inflammatory action of HSAT+ also protects against a chronic vascular-inflammatory disease, atherosclerosis, we conducted a human candidate-gene association study on >2000 coronary catheterization patients. Bioinformatic analysis of the HS3ST1 gene identified an intronic SNP, rs16881446, in a putative transcriptional regulatory region. The rs16881446G/G genotype independently associated with the severity of coronary artery disease and atherosclerotic cardiovascular events. In primary endothelial cells, the rs16881446G allele associated with reduced HS3ST1 expression. Together with the mouse data, this leads us to conclude that the HS3ST1 gene is required for AT's anti-inflammatory activity that appears to protect against acute and chronic inflammatory disorders.

Designing multivalent proteins based on natural killer cell receptors and their ligands as immunotherapy for cancer.

INTRODUCTION: Natural killer (NK) cells are an important component of the innate immune system that play a key role in host immunity against cancer. NK cell recognition and activation is based on cell surface receptors recognizing specific ligands that are expressed on many types of tumor cells. Some of these receptors are capable of activating NK cell function while other receptors inhibit NK cell function. Therapeutic approaches to treat cancer have been developed based on preventing NK cell inhibition or using NK cell receptors and their ligands to activate NK cells or T cells to destroy tumor cells. AREAS COVERED: This article describes the various strategies for targeting NK cell receptors and NK cell receptor ligands using multivalent proteins to activate immunity against cancer. EXPERT OPINION: NK cell receptors work in synergy to activate NK cell effector responses. Effective anti-cancer strategies will need to not only kill tumor cells but must also lead to the destruction of the tumor microenvironment. Immunotherapy based on NK cells and their receptors has the capacity to accomplish this through triggering lymphocyte cytotoxicity and cytokine production.

The diaphragms of fenestrated endothelia: gatekeepers of vascular permeability and blood composition.

Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.

SPECT imaging of peripheral amyloid in mice by targeting hyper-sulfated heparan sulfate proteoglycans with specific scFv antibodies.

INTRODUCTION: Amyloid deposits are associated with a broad spectrum of disorders including monoclonal gammopathies, chronic inflammation, and Alzheimer's disease. In all cases, the amyloid pathology contains, in addition to protein fibrils, a plethora of associated molecules, including high concentrations of heparan sulfate proteoglycans (HSPGs). METHODS: We have evaluated radioiodinated scFvs that bind HS for their ability to image amyloid deposits in vivo. scFv's with different binding characteristics were isolated by phage display using HS extracted from bovine kidney or mouse and human skeletal muscle glycosaminoglycans (GAGs). Following purification and radioiodination, the biodistribution of (125)I-scFv's was assessed in mice with inflammation-associated AA amyloidosis or in amyloid-free mice by using SPECT imaging, biodistribution measurements and tissue autoradiography. RESULTS: Four different scFv's all showed binding in vivo to amyloid in the spleen, liver and kidney of diseased mice; however, three of the scFv's also bound to sites within these organs in disease free mice. One scFv specific for hypersulfated HSPGs preferentially bound amyloid and did not accumulate in healthy tissues. CONCLUSIONS: These data indicate that HS expressed in amyloid deposits has unique qualities that can be distinguished from HS in normal tissues. A scFv specific for rare hypersulfated HS was used to selectively image AA amyloid in mice with minimal retention in normal tissue.

Anticoagulant heparan sulfate to not clot--or not?

Vascular endothelial cells (ECs) produce anticoagulant heparan sulfate (HSAT+)-a small subpopulation of heparan sulfate (HS) containing a specific pentasaccharide motif with high affinity for plasma antithrombin (AT). This pentasaccharide is responsible for the anticoagulant action of therapeutic heparin, which dramatically catalyzes AT neutralization of coagulation proteases. Consequently, HSAT+ has been designated as "anticoagulant HS," and has long been thought to convey antithrombotic properties to the blood vessel wall. The Hs3st1 gene encodes HS 3-O-sulfotransferase-1, whose rate limiting action regulates EC production of HSAT+. To elucidate the biologic role of HSAT+, we generated Hs3st1-/- knock-out mice that have undetectable EC HSAT+. Despite long held historic expectations, hemostasis was unaffected in Hs3st1-/- mice. In light of this surprising finding, herein we evaluate historic, biochemical, kinetic, physiologic, and molecular genetic studies of AT, heparin, and HSAT+. We find that a hemostatic role for HSAT+ cannot presently be excluded; however, HSAT+ may well not be essential for AT's anticoagulant function. Specifically, in the absence of glycosaminoglycans, physiologic levels of AT can neutralize coagulation proteases at a sufficiently high throughput to account for most of AT's anticoagulant function. Moreover, at the vessel wall surface, glycosaminoglycans distinct from HSAT+ may be the predominant catalysts of AT's anticoagulant activity. We then explore the possibility that HSAT+ regulates a less well known function of AT, anti-inflammatory activity. We find that Hs3st1-/- mice exhibit a strong proinflammatory phenotype that is unresponsive to AT's anti-inflammatory activity. We conclude that the predominant function of HSAT+ is to mediate AT's anti-inflammatory activity.

The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

Heparan sulfates in the lung: structure, diversity, and role in pulmonary emphysema.

There is an emerging interest in the extracellular matrix (ECM) of the lung, especially in the role it plays in development and disease. There is a rapid change from the classical view of the ECM as a supporting structure towards a view of the ECM as a regulatory entity with profound effects on proliferation, migration, and differentiation of pulmonary cells. In the ECM, a variety of molecules is present in a highly organized pattern. Next to the abundant fiber-forming molecules such as collagens and elastin, a large number of less abundant molecules are part of the ECM, including proteoglycans. In this review, we will focus on one class of proteoglycans, the heparan sulfate proteoglycans. We will particularly address the structure, biosynthesis, and function of their saccharide moiety, the heparan sulfates, including their role in development and (patho)physiology.

Aberrant fibrillin-1 expression in early emphysematous human lung: a proposed predisposition for emphysema.

Parenchymal destruction, airspace enlargement, and loss of elasticity are hallmarks of pulmonary emphysema. Although the basic mechanism is unknown, there is a consensus that malfunctioning of the extracellular matrix is a major contributor to the pathogenesis of emphysema. In this study, we analyzed the expression of the elastic fiber protein fibrillin-1 in a large number (n=69) of human lung specimens with early-onset emphysema. Specimens were morphologically characterized by the Destructive Index, the Mean Linear Intercept, and the Panel Grading. We observed a strong correlation (P<0.001) of aberrant fibrillin-1 staining with the degree of destruction of lung parenchyma (r=0.71), airspace enlargement (r=0.47), and emphysema-related morphological abnormalities (r=0.69). There were no obvious correlations with age and smoking behavior. Staining for three other extracellular matrix components (type I collagen, type IV collagen, and laminin) was not affected. The aberrant fibrillin-1 staining observed in this study is similar to that observed in Marfan syndrome, a syndrome caused by mutations in the gene encoding fibrillin-1. Strikingly, emphysema is noticed in a number of Marfan patients. This, together with the notion that disruption of the fibrillin-1 gene in mice results in emphysematous lesions, makes fibrillin-1 a strong candidate to be involved in the etiology and pathogenesis of emphysema.

Characterization of anticoagulant heparinoids by immunoprofiling.

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and-when additional saccharides are present-inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.

Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus.

The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.

Phage display-derived human antibodies against specific glycosaminoglycan epitopes.

Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hyaluronic acid, and keratan sulfate). Modification of the backbone by sulfation, deacetylation, and epimerization results in unique sequences within GAG molecules, which are instrumental in the binding of a large number of proteins. Investigating the exact roles of GAGs has long been hampered by the lack of appropriate tools, but we have successfully implemented phage display technology to generate a large panel of antibodies against CS, DS, HS, and heparin epitopes. These antibodies provide unique and highly versatile tools to study the topography, structure, and function of specific GAG domains. In this chapter, we describe the selection, characterization, and application of antibodies against specific GAG epitopes.

Heterogeneity of heparan sulfates in human lung.

Heparan sulfates (HS), a class of glycosaminoglycans, are long linear complex polysaccharides covalently attached to a protein core. The HS molecules are made up of repeating disaccharides onto which modification patterns are superimposed. This results in a large structural heterogeneity and forms the basis of specific interactions of HS toward a vast array of proteins, including growth factors and proteases. To study HS heterogeneity in the lung, we used phage display technology to select seven antibodies against human lung HS. Antibodies reacted with HS/heparin, but not with other glycosaminoglycans or polyanions. Sulfate groups were essential for antibody binding. The amino acid sequence of the antibodies was established, the complementarity determining region 3 of the heavy chain containing basic amino acids. The antibodies defined HS epitopes with a characteristic tissue distribution. Antibody EV3A1 primarily stained macrophages. Other antibodies primarily stained basement membranes, but with different preference toward type of basement membrane. Antibody EV3C3 was the only antibody which clearly reacted with bronchiolar epithelial cells. In human lung parenchyma, basic fibroblast growth factor and vascular endothelial growth factor were largely bound by HS. Some antibodies blocked a basic fibroblast growth factor-binding site of HS, and one antibody blocked a vascular endothelial growth factor-binding site of heparin. Taken together, these data suggest a specific role for HS epitopes in human lung. The antibodies obtained may be valuable tools to study HS in pulmonary diseases.