IL-33-binding HpARI family homologues with divergent effects in suppressing or enhancing type 2 immune responses.

HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains three HpARI family members and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar affinity for mouse IL-33; however, HpARI3 does not block IL-33-ST2 interactions. Instead, HpARI3 stabilizes IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together, these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and distinct functions, comprising a complex immunomodulatory arsenal of host-targeted proteins.

Breaking the Glyco-Code of HIV Persistence and Immunopathogenesis.

PURPOSE OF REVIEW: Glycoimmunology is an emerging field focused on understanding how immune responses are mediated by glycans (carbohydrates) and their interaction with glycan-binding proteins called lectins. How glycans influence immunological functions is increasingly well understood. In a parallel way, in the HIV field, it is increasingly understood how the host immune system controls HIV persistence and immunopathogenesis. However, what has mostly been overlooked, despite its potential for therapeutic applications, is the role that the host glycosylation machinery plays in modulating the persistence and immunopathogenesis of HIV. Here, we will survey four areas in which the links between glycan-lectin interactions and immunology and between immunology and HIV are well described. For each area, we will describe these links and then delineate the opportunities for the HIV field in investigating potential interactions between glycoimmunology and HIV persistence/immunopathogenesis. RECENT FINDINGS: Recent studies show that the human glycome (the repertoire of human glycan structures) plays critical roles in driving or modulating several cellular processes and immunological functions that are central to maintaining HIV infection. Understanding the links between glycoimmunology and HIV infection may create a new paradigm for discovering novel glycan-based therapies that can lead to eradication, functional cure, or improved tolerance of lifelong infection.

Galectin-3 interacts with the cell-surface glycoprotein CD146 (MCAM, MUC18) and induces secretion of metastasis-promoting cytokines from vascular endothelial cells.

The galactoside-binding protein galectin-3 is increasingly recognized as an important player in cancer development, progression, and metastasis via its interactions with various galactoside-terminated glycans. We have shown previously that circulating galectin-3, which is increased up to 30-fold in cancer patients, promotes blood-borne metastasis in an animal cancer model. This effect is partly attributable to the interaction of galectin-3 with unknown receptor(s) on vascular endothelial cells and causes endothelial secretion of several metastasis-promoting cytokines. Here we sought to identify the galectin-3-binding molecule(s) on the endothelial cell surface responsible for the galectin-3-mediated cytokine secretion. Using two different galectin-3 affinity purification processes, we extracted four cell membrane glycoproteins, CD146/melanoma cell adhesion molecule (MCAM)/MUC18, CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1), CD144/VE-cadherin, and CD106/Endoglin, from vascular endothelial cells. CD146 was the major galectin-3-binding ligand and strongly co-localized with galectin-3 on endothelial cell surfaces treated with exogenous galectin-3. Moreover, galectin-3 bound to N-linked glycans on CD146 and induced CD146 dimerization and subsequent activation of AKT signaling. siRNA-mediated suppression of CD146 expression completely abolished the galectin-3-induced secretion of IL-6 and G-CSF cytokines from the endothelial cells. Thus, CD146/MCAM is the functional galectin-3-binding ligand on endothelial cell surfaces responsible for galectin-3-induced secretion of metastasis-promoting cytokines. We conclude that CD146/MCAM interactions with circulating galectin-3 may have an important influence on cancer progression and metastasis.

TNF up-regulates ST3GAL4 and sialyl-Lewisx expression in lung epithelial cells through an intronic ATF2-responsive element.

We have previously shown that tumor necrosis factor (TNF) induced the up-regulation of the sialyltransferase gene ST3GAL4 (α2,3-sialyltransferase gene) BX transcript through mitogen- and stress-activated kinase 1/2 (MSK1/2), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. This up-regulation resulted in sialyl-Lewisx (sLex) overexpression on high-molecular-weight glycoproteins in inflamed airway epithelium and increased the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells. In the present study, we describe a TNF-responsive element in an intronic region of the ST3GAL4 gene, whose TNF-dependent activity is repressed by ERK/p38 and MSK1/2 inhibitors. This TNF-responsive element contains potential binding sites for ETS1 and ATF2 transcription factors related to TNF signaling. We also show that ATF2 is involved in TNF responsiveness, as well as in TNF-induced ST3GAL4 BX transcript and sLex overexpression in A549 lung epithelial cells. Moreover, we show that TNF induces the binding of ATF2 to the TNF-responsive element. Altogether, these data suggest that ATF2 could be a potential target to prevent inflammation-induced P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.

TNF induces the expression of the sialyltransferase ST3Gal IV in human bronchial mucosa via MSK1/2 protein kinases and increases FliD/sialyl-Lewis(x)-mediated adhesion of Pseudomonas aeruginosa.

We have shown previously that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLe(x) (sialyl-Lewis(x)) biosynthesis through the up-regulation of the BX transcript isoform of the ST3GAL4 (ST3 ß-galactoside α-2,3-sialyltransferase 4) sialyltransferase gene in lung epithelial cells and human bronchial mucosa. In the present study, we show that the TNF-induced up-regulation of the ST3GAL4 BX transcript is mediated by MSK1/2 (mitogen- and stress-activated kinase 1/2) through the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLe(x) expression on high-molecular-mass glycoproteins in inflamed airway epithelium. We also show that the TNF-induced sLe(x) expression increases the adhesion of the Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that ERK and p38 MAPK, and the downstream kinase MSK1/2, should be considered as potential targets to hamper inflammation, bronchial mucin glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.

Structural basis for IL-33 recognition and its antagonism by the helminth effector protein HpARI2.

IL-33 plays a significant role in inflammation, allergy, and host defence against parasitic helminths. The model gastrointestinal nematode Heligmosomoides polygyrus bakeri secretes the Alarmin Release Inhibitor HpARI2, an effector protein that suppresses protective immune responses and asthma in its host by inhibiting IL-33 signalling. Here we reveal the structure of HpARI2 bound to mouse IL-33. HpARI2 contains three CCP-like domains, and we show that it contacts IL-33 primarily through the second and third of these. A large loop which emerges from CCP3 directly contacts IL-33 and structural comparison shows that this overlaps with the binding site on IL-33 for its receptor, ST2, preventing formation of a signalling complex. Truncations of HpARI2 which lack the large loop from CCP3 are not able to block IL-33-mediated signalling in a cell-based assay and in an in vivo female mouse model of asthma. This shows that direct competition between HpARI2 and ST2 is responsible for suppression of IL-33-dependent responses.

Accumulation of unusual gangliosides G(Q3) and G(P3) in breast cancer cells expressing the G(D3) synthase.

Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward glycolipid substrates. ST8Sia I (EC 2.4.99.8, SAT-II, SIAT 8a) is the key enzyme controlling the biosynthesis of b- and c-series gangliosides. ST8Sia I is expressed at early developmental stages whereas in adult human tissues, ST8Sia I transcripts are essentially detected in brain. ST8Sia I together with b- and c-series gangliosides are also over-expressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, neuroblastoma and in estrogen receptor (ER) negative breast cancer, where they play a role in cell proliferation, migration, adhesion and angiogenesis. We have stably expressed ST8Sia I in MCF-7 breast cancer cells and analyzed the glycosphingolipid composition of wild type (WT) and GD3S+ clones. As shown by mass spectrometry, MCF-7 expressed a complex pattern of neutral and sialylated glycosphingolipids from globo- and ganglio-series. WT MCF-7 cells exhibited classical monosialylated gangliosides including G(M3), G(M2), and G(M1a). In parallel, the expression of ST8Sia I in MCF-7 GD3S+ clones resulted in a dramatic change in ganglioside composition, with the expression of b- and c-series gangliosides as well as unusual tetra- and pentasialylated lactosylceramide derivatives G(Q3) (II(3)Neu5Ac(4)-Gg(2)Cer) and G(P3) (II(3)Neu5Ac(5)-Gg(2)Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context.

Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Interferon-α alters host glycosylation machinery during treated HIV infection.

BACKGROUND: A comprehensive understanding of host factors modulated by the antiviral cytokine interferon-α (IFNα) is imperative for harnessing its beneficial effects while avoiding its detrimental side-effects during HIV infection. Cytokines modulate host glycosylation which plays a critical role in mediating immunological functions. However, the impact of IFNα on host glycosylation has never been characterized. METHODS: We assessed the impact of pegylated IFNα2a on IgG glycome, as well as CD8+ T and NK cell-surface glycomes, of 18 HIV-infected individuals on suppressive antiretroviral therapy. We linked these glycomic signatures to changes in inflammation, CD8+ T and NK cell phenotypes, and HIV DNA. FINDINGS: We identified significant interactions that support a model in which a) IFNα increases the proportion of pro-inflammatory, bisecting GlcNAc glycans (known to enhance FcγR binding) within the IgG glycome, which in turn b) increases inflammation, which c) leads to poor CD8+ T cell phenotypes and poor IFNα-mediated reduction of HIV DNA. Examining cell-surface glycomes, IFNα increases levels of the immunosuppressive GalNAc-containing glycans (T/Tn antigens) on CD8+ T cells. This induction is associated with lower HIV-gag-specific CD8+ T cell functions. Last, IFNα increases levels of fucose on NK cells. This induction is associated with higher NK functions upon K562 stimulation. INTERPRETATION: IFNα causes host glycomic alterations that are known to modulate immunological responses. These alterations are associated with both detrimental and beneficial consequences of IFNα. Manipulating host glycomic interactions may represent a strategy for enhancing the positive effects of IFNα while avoiding its detrimental side-effects. FUNDING: NIH grants R21AI143385, U01AI110434.

Sialyl-LewisX Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy.

A comprehensive understanding of the phenotype of persistent HIV-infected cells, transcriptionally active and/or transcriptionally inactive, is imperative for developing a cure. The relevance of cell-surface glycosylation to HIV persistence has never been explored. We characterize the relationship between cell-surface glycomic signatures and persistent HIV transcription in vivo. We find that the cell surface of CD4+ T cells actively transcribing HIV, despite suppressive therapy, harbors high levels of fucosylated carbohydrate ligands, including the cell extravasation mediator Sialyl-LewisX (SLeX), compared with HIV-infected transcriptionally inactive cells. These high levels of SLeX are induced by HIV transcription in vitro and are maintained after therapy in vivo. Cells with high-SLeX are enriched with markers associated with HIV susceptibility, signaling pathways that drive HIV transcription, and pathways involved in leukocyte extravasation. We describe a glycomic feature of HIV-infected transcriptionally active cells that not only differentiates them from their transcriptionally inactive counterparts but also may affect their trafficking abilities.

Galectin-9 Mediates HIV Transcription by Inducing TCR-Dependent ERK Signaling.

Endogenous plasma levels of the immunomodulatory carbohydrate-binding protein galectin-9 (Gal-9) are elevated during HIV infection and remain elevated after antiretroviral therapy (ART) suppression. We recently reported that Gal-9 regulates HIV transcription and potently reactivates latent HIV. However, the signaling mechanisms underlying Gal-9-mediated viral transcription remain unclear. Given that galectins are known to modulate T cell receptor (TCR)-signaling, we hypothesized that Gal-9 modulates HIV transcriptional activity, at least in part, through inducing TCR signaling pathways. Gal-9 induced T cell receptor ζ chain (CD3ζ) phosphorylation (11.2 to 32.1%; P = 0.008) in the J-Lat HIV latency model. Lck inhibition reduced Gal-9-mediated viral reactivation in the J-Lat HIV latency model (16.8-0.9%; P < 0.0001) and reduced both Gal-9-mediated CD4+ T cell activation (10.3 to 1.65% CD69 and CD25 co-expression; P = 0.0006), and IL-2/TNFα secretion (P < 0.004) in primary CD4+ T cells from HIV-infected individuals on suppressive ART. Using phospho-kinase antibody arrays, we found that Gal-9 increased the phosphorylation of the TCR-downstream signaling molecules ERK1/2 (26.7-fold) and CREB (6.6-fold). ERK and CREB inhibitors significantly reduced Gal-9-mediated viral reactivation (16.8 to 2.6 or 12.6%, respectively; P < 0.0007). Given that the immunosuppressive rapamycin uncouples HIV latency reversal from cytokine-associated toxicity, we also investigated whether rapamycin could uncouple Gal-9-mediated latency reactivation from its concurrent pro-inflammatory cytokine production. Rapamycin reduced Gal-9-mediated secretion of IL-2 (4.4-fold, P = 0.001) and TNF (4-fold, P = 0.02) without impacting viral reactivation (16.8% compared to 16.1%; P = 0.2). In conclusion, Gal-9 modulates HIV transcription by activating the TCR-downstream ERK and CREB signaling pathways in an Lck-dependent manner. Our findings could have implications for understanding the role of endogenous galectin interactions in modulating TCR signaling and maintaining chronic immune activation during ART-suppressed HIV infection. In addition, uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of recombinant Gal-9 within the reversal of HIV latency eradication framework.

Frontline Science: Plasma and immunoglobulin G galactosylation associate with HIV persistence during antiretroviral therapy.

Global antibody glycosylation is dynamic and plays critical roles in shaping different immunological outcomes and direct antibody functionality during HIV infection. However, the relevance of global antibody or plasma glycosylation patterns to HIV persistence after antiretroviral therapy (ART) has not been characterized. First, we compared glycomes of total plasma and isolated immunoglobulin G (IgG) from HIV+ ART-suppressed, HIV+ viremic, and HIV-negative individuals. Second, in ART-suppressed individuals, we examined the associations between glycomes and (1) levels of cell-associated HIV DNA and RNA in PBMCs and isolated CD4+ T cells, (2) CD4 count and CD4%, and (3) expression of CD4+ T-cell activation markers. HIV infection is associated with persistent alterations in the IgG glycome including decreased levels of disialylated glycans, which is associated with a lower anti-inflammatory activity, and increased levels of fucosylated glycans, which is associated with lower antibody-dependent cell-mediated cytotoxicity (ADCC). We also show that levels of certain mono- and digalactosylated nonfucosylated glycomic traits (A2G1, A2G2, and A2BG2), which have been reported to be associated with higher ADCC and higher anti-inflammatory activities, exhibit significant negative correlations with levels of cell-associated total HIV DNA and HIV RNA in ART-suppressed individuals. Finally, levels of certain circulating anti-inflammatory glycans are associated with higher levels of CD4 T cells and lower levels of T-cell activation. Our findings represent the first proof-of-concept evidence that glycomic alterations, known to be associated with differential states of inflammation and ADCC activities, are also associated with levels of HIV persistence in the setting of ART suppression.

Role of Cytokine-Induced Glycosylation Changes in Regulating Cell Interactions and Cell Signaling in Inflammatory Diseases and Cancer.

Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in number of diseases such as cancer and chronic inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases involved in the biosynthesis of carbohydrate chains. These changes in cell surface glycosylation are also known to regulate cell signaling and could contribute to disease pathogenesis. This review summarizes our current knowledge of the glycosylation changes induced by pro-inflammatory cytokines, with a particular focus on cancer and cystic fibrosis, and their consequences on cell interactions and signaling.

Estradiol represses the G(D3) synthase gene ST8SIA1 expression in human breast cancer cells by preventing NFκB binding to ST8SIA1 promoter.

Recent data have underlined a possible role of G(D3) synthase (GD3S) and complex gangliosides in Estrogen Receptor (ER) negative breast cancer progression. Here, we describe the main transcript of the GD3S coding gene ST8SIA1 expressed in breast tumors. We characterized the corresponding core promoter in Hs578T breast cancer cells and showed that estradiol decreases ST8SIA1 mRNA expression in ER-positive MCF-7 cells and ERα-transfected ER-negative Hs578T cells. The activity of the core promoter sequence of ST8SIA1 is also repressed by estradiol. The core promoter of ST8SIA1 contains two putative Estrogen Response Elements (ERE) that were not found to be involved in the promoter activity pathway. However, NFκB was shown to be involved in ST8SIA1 transcriptional activation and we demonstrated that estradiol prevents NFκB to bind to ST8SIA1 core promoter in ERα expressing breast cancer cells by inhibiting p65 and p50 nucleus localization. The activation of NFκB pathway in ER-negative tumors, due to the absence of estradiol signaling, might explain the overexpression of G(D3) synthase in this tumor subtype.

Over-sulfated glycosaminoglycans are alternative selectin ligands: insights into molecular interactions and possible role in breast cancer metastasis.

Distant metastasis account for about 90 % of cancer associated deaths, and yet the oncology field is cruelly lacking tools to accurately predict and/or prevent metastasis. Distant metastasis occurs when circulating tumor cells interact with the endothelium of distant organs and extravasate from the blood vessel into the surrounding tissue. Selectins are a family of carbohydrate receptors well depicted for their role in tumor cells extravasation. They mediate primary interactions of cancer cells with endothelial cells, as well as secondary interactions with leucocytes and platelets, which are also promoting metastasis. The cancer associated carbohydrate antigen sialyl-Lewis x (sLe(x)) has been repeatedly shown to be involved, as selectin ligand, in these interactions. However, recent studies have highlighted that glycosaminoglycans (GAGs), another class of glycans, may also serve as ligands for selectins. We report herein that cancer-associated GAGs are differentially recognized by selectins according to their density of sulfation and the pH conditions of the binding. We also show that these parameters regulate platelets-cancer cells heterotypic aggregation, supporting the idea that GAGs may have pro-metastatic function. Combining our experimental results with in depth analyses of molecular dockings, we propose a model of GAG/selectin interactions robust enough to recapitulate the differential binding of selectins to GAGs, the competition between GAGs and sLe(x) for selectin binding and the effect of sub-physiological pH on GAGs affinities towards selectins. Altogether, our data suggest GAGs to be good ligands for selectins, potentially promoting distant metastasis in a complementary way to sLe(x).

Sialyltransferases functions in cancers.

Abnormally elevated levels of sialylated tumor associated carbohydrate antigens are frequently described at the surface of cancer cells and/or secreted in biological fluids. It is now well established that this over-expression may result from deregulation in sialyltransferases enzymatic activity involved in their biosynthesis, but the precise molecular mechanisms remain unknown. Twenty different human sialyltransferases preside to the sialylation of glycoconjugates, either glycolipids or glycoproteins. This review summarizes the current knowledge on human sialyltransferases implicated in the altered expression of sialylated tumor associated antigens, the molecular basis of their regulated expression in cancer cells and the various tools developed by researchers and clinicians for their study in pathological samples.

TNF regulates sialyl-Lewisx and 6-sulfo-sialyl-Lewisx expression in human lung through up-regulation of ST3GAL4 transcript isoform BX.

Bronchial mucins from severely infected patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis exhibit increased amounts of sialyl-Lewis(x) (NeuAcα2-3Galß1-4[Fucα1-3]GlcNAc-R, sLe(x)) glycan structures. In cystic fibrosis, sLe(x) and its sulfated form 6-sulfo-sialyl-Lewis(x) (NeuAcα2-3Galß1-4[Fucα1-3](HO(3)S-6)GlcNAc-R, 6-sulfo-sLe(x)) serve as receptors for Pseudomonas aeruginosa and are involved in the chronicity of airway infection. However, little is known about the molecular mechanisms regulating the changes in glycosylation and sulfation of mucins in airways. Herein, we show that the pro-inflammatory cytokine TNF increases the expression of α2,3-sialyltransferase gene ST3GAL4, both in human bronchial mucosa and in A549 lung carcinoma cells. The role of sialyltransferase ST3Gal IV in sLe(x) biosynthesis was confirmed by siRNA silencing of ST3GAL4 gene. BX is the major transcript isoform expressed in healthy bronchial mucosa and in A549 cells, and is up-regulated by TNF in both models. Bioinformatics analysis and luciferase assays have confirmed that the 2 kb genomic sequence surrounding BX exon contains a promoter region regulated by TNF-related transcription factors. These results support further work aiming at the development of anti-inflammatory strategy to reduce chronic airway infection in diseases such as cystic fibrosis.