Abstract 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.
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.
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.
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%;
= 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%;
< 0.0001) and reduced both Gal-9-mediated CD4
T cell activation (10.3 to 1.65% CD69 and CD25 co-expression;
= 0.0006), and IL-2/TNFα secretion (
< 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;
< 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,
= 0.001) and TNF (4-fold,
= 0.02) without impacting viral reactivation (16.8% compared to 16.1%;
= 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.
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.
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-Lewis
(sLe
) 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 sLe
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.
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.
Display omitted
•Persistent HIV transcription modulates the glycosylation of infected cells•Cells actively transcribing HIV harbor high levels of fucosylated carbohydrates•HIV transcription induces the fucosylated extravasation mediator, Sialyl-LewisX•Cells with high Sialyl-LewisX are enriched with pathways involved in trafficking
Cell-surface glycans play a critical role in cell functions and fate. Nevertheless, the relevance of host glycosylation to HIV persistence is unknown. Colomb et al. characterized the cell-surface glycomes of HIV-infected cells during therapy and identified glycomic signatures of these cells that may affect cell trafficking and therefore HIV persistence.
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.
Bronchial mucins from severely infected patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis exhibit increased amounts of sialyl-Lewisx ...(NeuAcα2-3Galβ1-4Fucα1-3GlcNAc-R, sLex) glycan structures. In cystic fibrosis, sLex and its sulfated form 6-sulfo-sialyl-Lewisx (NeuAcα2-3Galβ1-4Fucα1-3(HO3S-6)GlcNAc-R, 6-sulfo-sLex) 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 sLex 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.
► TNF up-regulates the expression of (6-sulfo)-sLex in human bronchial mucosa. ► ST3Gal IV is critical for (6-sulfo)sLex biosynthesis in human lung. ► TNF induces the over-expression of the BX transcript of ST3GAL4 gene. ► The BX promoter region has been cloned and is responsive to TNF. ► TNF-induced ST3Gal IV expression could explain chronic infection of CF patients.
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 GM3, GM2, and GM1a. 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 GQ3 (II3Neu5Ac4-Gg2Cer) and GP3 (II3Neu5Ac5-Gg2Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context.
PDF
