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Changes in glycosylation during tumour progression are a key hallmark of cancer. One of the glycan moieties generally overexpressed in cancer are sialic acids, which can induce immunomodulatory properties via binding to Siglec receptors. We here show that Pancreatic Ductal Adenocarcinoma (PDAC) tumour cells present an increased sialylation that can be recognized by Siglec-7 and Siglec-9 on myeloid cells. We identified the expression of the α2,3 sialyltransferases ST3GAL1 and ST3GAL4 as main contributor to the synthesis of ligands for Siglec-7 and Siglec-9 in tumour cells. Analysing the myeloid composition in PDAC, using single cell and bulk transcriptomics data, we identified monocyte-derived macrophages as contributors to the poor clinical outcome. Tumour-derived sialic acids dictate monocyte to macrophage differentiation via signalling through Siglec-7 and Siglec-9. Moreover, triggering of Siglec-9 in macrophages reduce inflammatory programmes, while increasing PD-L1 and IL-10 expression, illustrating that sialic acids modulate different myeloid cells. This work highlights a critical role for sialylated glycans in controlling immune suppression and provides new potential targets for cancer immunotherapy in PDAC. Alterations in glycosylation in tumours facilitate tumour progression. Here, the authors show that pancreatic ductal adenocarcinomas present increased sialylation, which stimulates the polarisation of monocytes via Siglec receptors, resulting in the generation of immune suppressive tumour associated macrophages.

Sialic acid-containing glycans (sialoglycans) on pathological cells interact with Siglecs, glyco-immune checkpoint receptors expressed on myeloid cells, suppressing the cytotoxic functions of these immune cells. Using targeted glycomic analyses and gene editing, we show that HIV infection reprograms the glycosylation machinery of infected cells to increase the expression of the sialoglycan ligands for Siglec-3, −7, and −9. These ligands engage Siglecs on myeloid cells, impairing their ability to target HIV-infected cells. Selective disruption of these interactions using 10-1074-SiaD, an HIV-specific antibody conjugated to sialidase, an enzyme that removes sialic acids, significantly enhances monocyte- and neutrophil-mediated killing of HIV-infected cells in autologous assays. Treatment with 10-1074-SiaD in female humanized mice infected with HIV reduces viral load and decreases inflammation. These findings reveal an immune evasion mechanism exploited by HIV to evade myeloid cell immune surveillance and highlight the potential of targeting sialoglycan-Siglec interactions to improve immune clearance of HIV-infected cells. Immune evasion mechanisms of initial HIV infection are incompletely understood. Here, the authors show that HIV rewires the glycosylation machinery of infected myeloid cells, forming a glycan shield that engages glyco-immune checkpoints and inhibits cell function, and thus targeted killing of infected cells.

Polysialic acid (polySia) emerges as a novel regulator of microglia activity. We recently identified polysialylated proteins in the Golgi compartment of murine microglia that are released in response to inflammatory stimulation. Since exogenously added polySia is able to attenuate the inflammatory response, we proposed that the release of polysialylated proteins constitutes a mechanism for negative feedback regulation of microglia activation. Here, we demonstrate that translocation of polySia from the Golgi to the cell surface can be induced by calcium depletion of the Golgi compartment and that polysialylated proteins are continuously released for at least 24 h after the onset of inflammatory stimulation. The latter was unexpected, because polySia signals detected by immunocytochemistry are rapidly depleted. However, it indicates that the amount of released polySia is much higher than anticipated based on immunostaining. This may be crucial for microglial responses during traumatic brain injury (TBI), as we detected polySia signals in activated microglia around a stab wound in the adult mouse brain. In BV2 microglia, the putative polySia receptor Siglec-E is internalized during lipopolysaccharide (LPS)-induced activation and in response to polySia exposure, indicating interaction. Correspondingly, CRISPR/Cas9-mediated Siglec-E knockout prevents inhibition of pro inflammatory activation by exogenously added polySia and leads to a strong increase of the LPS response. A comparable increase of LPS-induced activation has been observed in microglia with abolished polySia synthesis. Together, these results indicate that the release of the microglia-intrinsic polySia pool, as implicated in TBI, inhibits the inflammatory response by acting as a trans-activating ligand of Siglec-E.

Bacterial vaginosis (BV) is a common vaginal infection that affects a significant proportion of women and is associated with reduced fertility and increased risk of secondary infections. Gardnerella vaginalis is the most well-known BV-associated bacterium, but Prevotella species including P. timonensis and P. bivia may also play an important role. We showed that, similar to G. vaginalis , P. timonensis adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria, P. timonensis was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV.

Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid–Siglec interactions between glioma cells and MDSCs in the tumor microenvironment.

Aberrant glycosylation, especially sialylation, on cell surface is often associated with cancer progression and immunosuppression. Over-sialylation of stage-specific embryonic antigen-4 (SSEA-4) to generate disialylGb5 (DSGb5) was reported to trigger Siglec-7 recognition and suppress NK-mediated target killing. In this study, efficient chemo-enzymatic and programmable one-pot methods were explored for the synthesis of DSGb5 and related sialosides for assembly of glycan microarrays and evaluation of binding specificity toward Siglecs-7, 9, 10, and 15 associated with immune checkpoint inhibition. The result showed weak binding of DSGb5 to these Siglecs; however, a truncated glycolyl glycan was identified to bind Siglec-10 strongly with a dissociation constant of 50 nM and exhibited a significant inhibition of Siglec-10 interacting with breast cancer cells.

Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent “molecular mimicry” of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic “Trojan horse” that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens. IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of “self” cells by the immune system, thereby guiding an appropriate immune response against foreign “nonself” and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called “Kdn” (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of receptors that recognize sialoglycans – sialic acid containing glycans that are abundantly present on cell membranes. Siglecs are expressed on most immune cells and can modulate their activity and function. The majority of Siglecs contains immune inhibitory motifs comparable to the immune checkpoint receptor PD-1. In the tumor microenvironment (TME), signaling through the Siglec-sialoglycan axis appears to be enhanced through multiple mechanisms favoring tumor immune evasion similar to the PD-1/PD-L1 signaling pathway. Siglec expression on tumor-infiltrating immune cells appears increased in the immune suppressive microenvironment. At the same time, enhanced Siglec ligand expression has been reported for several tumor types as a result of aberrant glycosylation, glycan modifications, and the increased expression of sialoglycans on proteins and lipids. Siglec signaling has been identified as important regulator of anti-tumor immunity in the TME, but the key factors contributing to Siglec activation by tumor-associated sialoglycans are diverse and poorly defined. Among others, Siglec activation and signaling are co-determined by their expression levels, cell surface distribution, and their binding preferences for cis- and trans -ligands in the TME. Siglec binding preference are co-determined by the nature of the proteins/lipids to which the sialoglycans are attached and the multivalency of the interaction. Here, we review the current understanding and emerging conditions and factors involved in Siglec signaling in the TME and identify current knowledge gaps that exist in the field.

Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody–sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody–sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy. An αHER2 antibody–neuraminidase conjugate, which selectively targets the removal of sialic acids from glycans on breast cancer cells, bypasses a glycoimmune checkpoint and enhances tumor cell killing by the host immune system.

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often lethal respiratory illness in humans, and no vaccines or specific treatments are available. Infections are initiated via binding of the MERS-CoV spike (S) glycoprotein to sialosides and dipeptidyl-peptidase 4 (the attachment and entry receptors, respectively). To understand MERS-CoV engagement of sialylated receptors, we determined the cryo-EM structures of S in complex with 5-N-acetyl neuraminic acid, 5-N-glycolyl neuraminic acid, sialyl-LewisX, α2,3-sialyl-N-acetyl-lactosamine and α2,6-sialyl-N-acetyl-lactosamine at 2.7–3.0 Å resolution. We show that recognition occurs via a conserved groove that is essential for MERS-CoV S-mediated attachment to sialosides and entry into human airway epithelial cells. Our data illuminate MERS-CoV S sialoside specificity and suggest that selectivity for α2,3-linked over α2,6-linked receptors results from enhanced interactions with the former class of oligosaccharides. This study provides a structural framework explaining MERS-CoV attachment to sialoside receptors and identifies a site of potential vulnerability to inhibitors of viral entry.