Siglec-F+ neutrophils in the spleen induce immunosuppression following acute infection.

Background: The mechanisms underlying the increased mortality of secondary infections during the immunosuppressive phase of sepsis remain elusive. Objectives: We sought to investigate the role of Siglec-F+ neutrophils on splenic T lymphocytes in the immunosuppressed phase of sepsis and on secondary infection in PICS mice, and to elucidate the underlying mechanisms. Methods: We established a mouse model of sepsis-induced immunosuppression followed by secondary infection with LPS or E. coli. The main manifestation of immunosuppression is the functional exhaustion of splenic T lymphocytes. Treg depletion reagent Anti-IL-2, IL-10 blocker Anti-IL-10R, macrophage depletion reagent Liposomes, neutrophil depletion reagent Anti-Ly6G, neutrophil migration inhibitor SB225002, Siglec-F depletion reagent Anti-Siglec-F are all used on PICS mice. The function of neutrophil subsets was investigated by adoptive transplantation and the experiments in vitro. Results: Compared to other organs, we observed a significant reduction in pro-inflammatory cytokines in the spleen, accompanied by a marked increase in IL-10 production, primarily by infiltrating neutrophils. These infiltrating neutrophils in the spleen during the immunosuppressive phase of sepsis undergo phenotypic change in the local microenvironment, exhibiting high expression of neutrophil biomarkers such as Siglec-F, Ly6G, and Siglec-E. Depletion of neutrophils or specifically targeting Siglec-F leads to enhance the function of T lymphocytes and a notable improvement in the survival of mice with secondary infections. Conclusions: We identified Siglec-F+ neutrophils as the primary producers of IL-10, which significantly contributed to T lymphocyte suppression represents a novel finding with potential therapeutic implications.

Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

Identification of BACH1-IT2-miR-4786-Siglec-15 immune suppressive axis in bladder cancer.

The sialic acid binding Ig like lectin 15 (Siglec-15) was previously identified as tumor immune suppressor gene in some human cancers with elusive molecular mechanism to be elucidated. The continuous focus on both clinical and basic biology of bladder cancer leads us to characterize aberrant abundance of BACH1-IT2 associating with stabilization of Siglec-15, which eventually contributes to local immune suppressive microenvironment and therefore tumor advance. This effect was evidently mediated by miR-4786-5p. BACH1-IT2 functions in this scenario as microRNA sponge, and competitively conceals miR-4786 and up-regulates cancer cell surface Siglec-15. The BACH1-IT2-miR-4786-Siglec-15 axis significantly influences activation of immune cell co-culture. In summary, our data highlights the critical involvements of BACH1-IT2 and miR-4786 in immune evasion in bladder cancer, which hints the potential for both therapeutic and prognostic exploitation.

Siglec9 + tumor-associated macrophages predict prognosis and therapeutic vulnerability in patients with colon cancer.

BACKGROUND: Siglec9 has been identified as an immune checkpoint molecule on tumor-associated macrophages (TAMs). Nevertheless, the expression profile and clinical significance of Siglec9 + TAMs in colon cancer (CC) are still not fully understood. METHODS: Two clinical cohorts from distinct medical centers were retrospectively enrolled. Immunohistochemistry and immunofluorescence were conducted to evaluate the infiltration of immune cells. Single-cell RNA sequencing and flow cytometry were utilized to identify the impact of Siglec9 + TAMs on the tumor immune environment, which was subsequently validated through bioinformatics analysis of the TCGA database. Prognosis and the benefit of adjuvant chemotherapy (ACT) were also evaluated using Cox regression analysis and the Kaplan-Meier method. RESULTS: High infiltration of Siglec9 + TAMs was associated with worse prognosis and better benefit from 6-month ACT. Siglec9 + TAMs contributed to immunoevasion by promoting the infiltration of immunosuppressive cells and the dysfunction process of CD8 + T cells. Additionally, high infiltration of Siglec9 + TAMs was associated with the mesenchymal-featured subtype and overexpression of the VEGF signaling pathway, which was validated by the strongest communication between Siglec9 + TAMs and vascular endothelial cells. CONCLUSIONS: Siglec9 + TAMs may serve as a biomarker for prognosis and response to ACT in CC. Furthermore, the immunoevasive contexture and angiogenesis stimulated by Siglec9 + TAMs suggest potential treatment combinations for CC patients.

Bacterial pseudaminic acid binding to Siglec-10 induces a macrophage interleukin-10 response and suppresses phagocytosis.

Pseudaminic acid (Pse) on pathogenic bacteria exopolysaccharide engages with the sialic acid-binding immunoglobulin-type lectin (Siglec)-10 receptor on macrophages via the critical 7-N-acetyl group. This binding stimulates macrophages to secrete interleukin 10 that suppresses phagocytosis against bacteria, but can be reverted by blocking Pse-Siglec-10 interaction with Pse-binding protein as a promising therapy.

Increased Siglec-9/Siglec-9L interactions on NK cells predict poor HCC prognosis and present a targetable checkpoint for immunotherapy.

BACKGROUND & AIMS: Natural killer (NK) cell-based anti-hepatocellular carcinoma (HCC) therapy is an increasingly attractive approach that warrants further study. Siglec-9 interacts with its ligand (Siglec-9L) and restrains NK cell functions, suggesting it is a potential therapeutic target. However, in situ Siglec-9/Siglec-9L interactions in HCC have not been reported, and a relevant interventional strategy is lacking. Herein, we aim to illustrate Siglec-9/Siglec-9L-mediated cell sociology and identify small-molecule inhibitors targeting Siglec-9 that could improve the efficacy of NK cell-based immunotherapy for HCC. METHODS: Multiplexed immunofluorescence staining was performed to analyze the expression pattern of Siglec-7, -9 and their ligands in HCC tissues. Then we conducted docking-based virtual screening combined with bio-layer interferometry assays to identify a potent small-molecule Siglec-9 inhibitor. The therapeutic potential was further evaluated in vitro and in hepatoma-bearing NCG mice. RESULTS: Siglec-9 expression, rather than Siglec-7, was markedly upregulated on tumor-infiltrating NK cells, which correlated significantly with reduced survival of patients with HCC. Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival, further suggesting that Siglec-9/Siglec-9L interactions are a potential therapeutic target in HCC. In addition, we identified a small-molecule Siglec-9 inhibitor MTX-3937 which inhibited phosphorylation of Siglec-9 and downstream SHP1 and SHP2. Accordingly, MTX-3937 led to considerable improvement in NK cell function. Notably, MTX-3937 enhanced cytotoxicity of both human peripheral and tumor-infiltrating NK cells. Furthermore, transfer of MTX-3937-treated NK92 cells greatly suppressed the growth of hepatoma xenografts in NCG mice. CONCLUSIONS: Our study provides the rationale for HCC treatment by targeting Siglec-9 on NK cells and identifies a promising small-molecule inhibitor against Siglec-9 that enhances NK cell-mediated HCC surveillance. IMPACT AND IMPLICATIONS: Herein, we found that Siglec-9 expression is markedly upregulated on tumor-infiltrating natural killer (TINK) cells and correlates with reduced survival in patients with hepatocellular carcinoma (HCC). Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival. More importantly, we identified a small-molecule inhibitor targeting Siglec-9 that augments NK cell functions, revealing a novel immunotherapy strategy for liver cancer that warrants further clinical investigation.

Expression of Siglec-9 in peripheral blood neutrophils was increased and associated with disease severity in patients with AECOPD.

BACKGROUND: The pathogenesis and treatment strategies for chronic obstructive pulmonary disease (COPD) require further exploration. Abnormal neutrophil inflammation and the overexpression of neutrophil extracellular traps (NETs) are closely associated with acute exacerbations of COPD (AECOPD). Siglec-9, a specific receptor expressed on neutrophils that inhibits their function, prompted us to investigate its relationship with NETs found in induced sputum and the severity of the disease. METHODS: We collected clinical data from patients with AECOPD and assessed the expression of Siglec-9 in peripheral blood neutrophils and the presence of NETs in induced sputum. We then observed the correlation between Siglec-9, the inflammatory response, and the severity of AECOPD. RESULTS: We observed an increase in the expression of Siglec-9 in the peripheral blood neutrophils of patients with AECOPD. Concurrently, these patients exhibited more severe clinical symptoms, higher systemic inflammation levels, and a reduced quality of life compared to those with induced sputum NET expression. Further subgroup analysis of AECOPD patients with high Siglec-9 expression revealed worsened quality of life and more severe inflammation, particularly in indicators such as the BODE index, CRP, peripheral blood neutrophil count, IL-6, IL-8, TNF-α expression, and others. Furthermore, we noted a significant increase in NET-specific expression in the sputum of patients with high Siglec-9 expression levels. In comparison to patients with low Siglec-9 expression, those with high expression experienced more systemic inflammatory reactions and a lower quality of life. Correlation analysis of the aforementioned indicators revealed that the expression ratio of Siglec-9 in the peripheral blood of patients correlated with lung function, quality of life, and NETs in the induced sputum of patients with AECOPD. CONCLUSION: The increased expression of Siglec-9 in peripheral blood neutrophils of AECOPD patients leads to elevated NET expression in induced sputum, exacerbating the systemic inflammatory response and worsening lung function and quality of life in these patients.

Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling.

Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G ß-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G' loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B'-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.

Aberrant GPA expression and regulatory function of red blood cells in sickle cell disease.

ABSTRACT: Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.

The sialic acid-Siglec immune checkpoint: an opportunity to enhance immune responses and therapy effectiveness in melanoma.

Modulation of immune responses through immune checkpoint blockade has revolutionized cutaneous melanoma treatment. However, it is still the case that not all patients respond successfully to these therapies, indicating the presence of as yet unknown resistance mechanisms. Hence, it is crucial to find novel targets to improve therapy efficacy. One of the described resistance mechanisms is regulated by immune inhibitory Siglec receptors, which are engaged by the carbohydrates sialic acids expressed on tumour cells, contributing to programmed cell death protein-1 (PD1)-like immune suppression mechanisms. In this review, we provide an overview on the regulation of sialic acid synthesis, its expression in melanoma, and the contribution of the sialic acid-Siglec axis to tumour development and immune suppressive mechanisms in the tumour microenvironment. Finally, we highlight potential sialic acid-Siglec axis-related therapeutics to improve the treatment of melanoma.

Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

Delivery of SIRT1 by cancer-associated adipocyte-derived extracellular vesicles regulates immune response and tumorigenesis of ovarian cancer cells.

PURPOSE: This study intends to investigate the possible molecular mechanism of immune response and tumorigenesis in ovarian cancer cells, mediated by sirtuin 1 (SIRT1)-containing extracellular vesicles (EVs) derived from cancer-associated adipocytes (CAAs) (CAA-EVs). METHODS: Differentially expressed genes in EVs from CAAs were screened by RNA transcriptome sequencing, and the downstream pathway was predicted in silico. The binding between SIRT1 and CD24 was investigated by luciferase activity and ChIP-PCR assays. EVs were extracted from human ovarian cancer tissue-isolated CAAs, and the internalization of CCA-EVs by ovarian cancer cells was characterized. The ovarian cancer cell line was injected into mice to establish an animal model. Flow cytometry was performed to analyze the proportions of M1 and M2 macrophages, CD8+ T, T-reg, and CD4+ T cells. TUNEL staining was used to detect cell apoptosis in the mouse tumor tissues. ELISA detection was performed on immune-related factors in the serum of mice. RESULTS: CAA-EVs could deliver SIRT1 to ovarian cancer cells, thereby affecting the immune response of ovarian cancer cells in vitro and promoting tumorigenesis in vivo. SIRT1 could transcriptionally activate the expression of CD24, and CD24 could up-regulate Siglec-10 expression. CAA-EVs-SIRT1 activated the CD24/Siglec-10 axis and promoted CD8+ T cell apoptosis, thereby promoting tumorigenesis in mice. CONCLUSION: CAA-EVs-mediated transfer of SIRT1 regulates the CD24/Siglec-10 axis to curb immune response and promote tumorigenesis of ovarian cancer cells.

Mast cell silencing: A novel therapeutic approach for urticaria and other mast cell-mediated diseases.

Chronic urticaria (CU) is a mast cell (MC)-dependent disease with limited therapeutic options. Current management strategies are directed at inhibiting IgE-mediated activation of MCs and antagonizing effects of released mediators. Due to the complexity and heterogeneity of CU and other MC diseases and mechanisms of MC activation-including multiple activating receptors and ligands, diverse signaling pathways, and a menagerie of mediators-strategies of MC depletion or MC silencing (i.e., inhibition of MC activation via binding of inhibitory receptors) have been developed to overcome limitations of singularly targeted agents. MC silencers, such as agonist monoclonal antibodies that engage inhibitory receptors (e.g., sialic acid-binding immunoglobulin-like lectin8 -[Siglec-8] [lirentelimab/AK002], Siglec-6 [AK006], and CD200R [LY3454738]), have reached preclinical and clinical stages of development. In this review, we (1) describe the role of MCs in the pathogenesis of CU, highlighting similarities with other MC diseases in disease mechanisms and response to treatment; (2) explore current therapeutic strategies, categorized by nonspecific immunosuppression, targeted inhibition of MC activation or mediators, and targeted modulation of MC activity; and (3) introduce the concept of MC silencing as an emerging strategy that could selectively block activation of MCs without eliciting or exacerbating on- or off-target, immunosuppressive adverse effects.

Siglec-E Ligand Downregulation on Hippocampus Neurons Induced Inflammation in Sevoflurane-Associated Perioperative Neurocognitive Disorders in Aged Mice.

Activated microglia-induced inflammation in the hippocampus plays an important role in perioperative neurocognitive disorders. Previous studies have shown that sialic acid-binding immunoglobulin-like lectin 3 (hSiglec-3, ortholog of mouse Siglec-E) engagement in microglia and its glycan ligands on neurons contributes to inflammatory homeostasis through an endogenous negative regulation pathway. This study aimed to explore whether the glycan ligand alteration on neurons plays a role in sevoflurane-induced perioperative neurocognitive disorders. This study's data has shown that a slight Siglec-E ligands' expression decrease does not induce inflammation homeostasis disruption. We also demonstrated that the ligand level on neurons was decreased with age, and the reduced Siglec-E ligand expression on neurons caused via sevoflurane was induced by neuraminidase 1. Furthermore, this study has shown that the Siglec-E ligand expression decline caused by age and sevoflurane treatment could decrease the ligands' level, thus leading to inflammatory homeostasis disruption. This research provided a novel mechanism for perioperative neurocognitive disorder susceptibility in the elderly.

Targeting myeloid cells for cancer immunotherapy: Siglec-7/9/10/15 and their ligands.

Advances in immunotherapy have revolutionized cancer treatment, yet many patients do not show clinical responses. While most immunotherapies target T cells, myeloid cells are the most abundant cell type in solid tumors and are key orchestrators of the immunosuppressive tumor microenvironment (TME), hampering effective T cell responses. Therefore, unraveling the immune suppressive pathways within myeloid cells could unveil new avenues for cancer immunotherapy. Over the past decade, Siglec receptors and their ligand, sialic acids, have emerged as a novel immune checkpoint on myeloid cells. In this review, we highlight key findings on how sialic acids modify immunity in the TME through engagement of Siglec-7/9/10/15 expressed on myeloid cells, and how the sialic acid-Siglec axis can be targeted for future cancer immunotherapies.

Detection of Intestinal Inflammation by Vascular Adhesion Protein-1-Targeted [68Ga]Ga-DOTA-Siglec-9 Positron Emission Tomography in Murine Models of Inflammatory Bowel Disease.

PURPOSE: Inflammatory bowel disease (IBD) can be imaged with positron emission tomography (PET), but existing PET radiopharmaceuticals have limited diagnostic accuracy. Vascular adhesion protein-1 (VAP-1) is an endothelial cell surface molecule that controls leukocyte extravasation into sites of inflammation. However, the role of inflammation-induced VAP-1 expression in IBD is still unclear. Therefore, this study investigated the utility of VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 positron emission tomography/computed tomography (PET/CT) for assessing inflammation in two mouse models of IBD. PROCEDURES: Studies were performed using K8-/- mice that develop a chronic colitis-phenotype and C57Bl/6NCrl mice with acute intestinal inflammation chemically-induced using 2.5% dextran sodium sulfate (DSS) in drinking water. In both diseased and control mice, uptake of the VAP-1-targeting peptide [68Ga]Ga-DOTA-Siglec-9 was assessed in intestinal regions of interest using in vivo PET/CT, after which ex vivo gamma counting, digital autoradiography, and histopathological analyses were performed. Immunofluorescence staining was performed to determine VAP-1-expression in the intestine, including in samples from patients with ulcerative colitis. RESULTS: Intestinal inflammation could be visualized by [68Ga]Ga-DOTA-Siglec-9 PET/CT in two murine models of IBD. In both models, the in vivo PET/CT and ex vivo studies of [68Ga]Ga-DOTA-Siglec-9 uptake were significantly higher than in control mice. The in vivo uptake was increased on average 1.4-fold in the DSS model and 2.0-fold in the K8-/- model. Immunofluorescence staining revealed strong expression of VAP-1 in the inflamed intestines of both mice and patients. CONCLUSIONS: This study suggests that the VAP-1-targeting [68Ga]Ga-DOTA-Siglec-9 PET tracer is a promising tool for non-invasive imaging of intestinal inflammation. Future studies in patients with IBD and evaluation of the potential value of [68Ga]Ga-DOTA-Siglec-9 in diagnosis and monitoring of the disease are warranted.

Quantitative Analysis of Siglec Ligands by Flow Cytometry.

Siglecs (sialic acid-binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor-type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi-quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec-Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec-Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec-Fc fusion protein Support Protocol 1: Sialidase treatment of mammalian cells Support Protocol 2: Preparation of recombinant Siglec-Fc fusion protein via transient transfection of mammalian cells.

SIGLEC-5/14 Inhibits CD11b/CD18 Integrin Activation and Neutrophil-Mediated Tumor Cell Cytotoxicity.

Since the successful introduction of checkpoint inhibitors targeting the adaptive immune system, monoclonal antibodies inhibiting CD47-SIRPα interaction have shown promise in enhancing anti-tumor treatment efficacy. Apart from SIRPα, neutrophils express a broad repertoire of inhibitory receptors, including several members of the sialic acid-binding receptor (SIGLEC) family. Here, we demonstrate that interaction between tumor cell-expressed sialic acids and SIGLEC-5/14 on neutrophils inhibits antibody-dependent cellular cytotoxicity (ADCC). We observed that conjugate formation and trogocytosis, both essential processes for neutrophil ADCC, were limited by the sialic acid-SIGLEC-5/14 interaction. During neutrophil-tumor cell conjugate formation, we found that inhibition of the interaction between tumor-expressed sialic acids and SIGLEC-5/14 on neutrophils increased the CD11b/CD18 high affinity conformation. By dynamic acoustic force measurement, the binding between tumor cells and neutrophils was assessed. The interaction between SIGLEC-5/14 and the sialic acids was shown to inhibit the CD11b/CD18-regulated binding between neutrophils and antibody-opsonized tumor cells. Moreover, the interaction between sialic acids and SIGLEC-5/14-consequently hindered trogocytosis and tumor cell killing. In summary, our results provide evidence that the sialic acid-SIGLEC-5/14 interaction is an additional target for innate checkpoint blockade in the tumor microenvironment.

[Research progress on the role and clinical application of Siglecs in tumor immunity].

Lectins are proteins responsible for recognizing the signals of sugar molecules in the body. Sialic acid-binding immunoglobulin-like lectins (Siglecs) regulate the innate and adaptive immune responses in the tumor microenvironment by recognizing the glycan structure containing sialic acid and mediating downstream signals through immune receptor tyrosine inhibitory motifs. In recent years, a variety of tumor treatment strategies targeting the sialic acid-Siglecs axis have been introduced, including sialoglycoprotein-mediated drug delivery and antibody mediated inhibition of Siglecs from recognizing tumor surface ligands. In the future, by combining with glycoprotein nanotherapy, antibody therapy and gene therapy, Siglecs can be used to accurately locate tumor targets and release the anti-tumor immunity, so as to achieve the purpose of effective cure of tumors.

Siglec-7 glyco-immune binding mAbs or NK cell engager biologics induce potent antitumor immunity against ovarian cancers.

Ovarian cancer (OC) is a lethal gynecologic malignancy, with modest responses to CPI. Engagement of additional immune arms, such as NK cells, may be of value. We focused on Siglec-7 as a surface antigen for engaging this population. Human antibodies against Siglec-7 were developed and characterized. Coculture of OC cells with PBMCs/NKs and Siglec-7 binding antibodies showed NK-mediated killing of OC lines. Anti-Siglec-7 mAb (DB7.2) enhanced survival in OC-challenged mice. In addition, the combination of DB7.2 and anti-PD-1 demonstrated further improved OC killing in vitro. To use Siglec-7 engagement as an OC-specific strategy, we engineered an NK cell engager (NKCE) to simultaneously engage NK cells through Siglec-7, and OC targets through FSHR. The NKCE demonstrated robust in vitro killing of FSHR+ OC, controlled tumors, and improved survival in OC-challenged mice. These studies support additional investigation of the Siglec-7 targeting approaches as important tools for OC and other recalcitrant cancers.