{"gene":"SIGLEC9","run_date":"2026-04-28T20:42:07","timeline":{"discoveries":[{"year":2000,"finding":"Siglec-9 is a type I transmembrane protein with three extracellular Ig-like domains (N-terminal V-set and two C2-set domains), a transmembrane region, and a cytoplasmic tail containing two tyrosine-based signaling motifs (one ITIM). Expression of full-length cDNA in COS cells induces sialic-acid-dependent erythrocyte binding. Recombinant soluble extracellular domain binds α2-3 and α2-6-linked sialic acids; mutation of a critical arginine in domain 1 abrogates binding.","method":"cDNA cloning, COS cell expression, erythrocyte binding assay, recombinant protein binding assay, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in heterologous cells, mutagenesis of active-site arginine, multiple orthogonal binding assays","pmids":["10801860"],"is_preprint":false},{"year":2000,"finding":"Siglec-9 is expressed at high or intermediate levels on monocytes, neutrophils, and a minor CD16+/CD56- population; weaker expression on ~50% of B cells and NK cells and minor CD8+ and CD4+ T cell subsets. Binding assays confirmed recognition of sialic acid in α2,3- or α2,6-glycosidic linkage to galactose.","method":"Flow cytometry with specific mAb, binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct flow cytometry localization plus binding assays, replicated across multiple labs","pmids":["10801862"],"is_preprint":false},{"year":2001,"finding":"The C-C' loop region (residues Asn70–Lys75) in the V-set sugar-binding domain determines the differential glycan binding specificities of Siglec-7 vs. Siglec-9. Siglec-9 binds LSTc and GD1a oligosaccharides but not GD3 and LSTb, whereas Siglec-7 shows the opposite preference. Substituting this region between the two siglecs swaps their binding specificities.","method":"Chimeric V-set domain mutagenesis, polyvalent glyco-probe binding assays on CHO cells, molecular modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — domain swap mutagenesis combined with structural modeling and glycan binding assays","pmids":["11741958"],"is_preprint":false},{"year":2004,"finding":"Siglec-9 negatively regulates T cell receptor (TCR) signaling: upon TCR engagement or pervanadate stimulation, Siglec-9 undergoes tyrosine phosphorylation and recruits SHP-1, reduces phosphorylation of ZAP-70 at Tyr319, and decreases NFAT transcriptional activity. Mutation of the conserved Arg120 in the ligand-binding site reduces inhibitory function, demonstrating that sialic acid ligand binding is required for optimal inhibition.","method":"Stable/transient transfection of Jurkat T cells, TCR stimulation assays, SHP-1 co-immunoprecipitation, NFAT/luciferase reporter, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — reconstitution in Jurkat cells, mutagenesis of ligand-binding arginine, multiple downstream readouts","pmids":["15292262"],"is_preprint":false},{"year":2005,"finding":"Siglec-9 ligation on neutrophils initiates two death pathways: (1) apoptotic (ROS- and caspase-dependent) under normal conditions, and (2) nonapoptotic/caspase-independent death (characterized by cytoplasmic vacuolization and ROS-dependence) when neutrophils are primed with proinflammatory cytokines (GM-CSF, IFN-α, IFN-γ). ROS scavengers and ROS-deficient neutrophils block both pathways.","method":"Siglec-9 ligation on primary neutrophils, caspase inhibitor assays, ROS scavenger experiments, ROS-deficient patient neutrophils, morphological analysis","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (pharmacological inhibition, genetic deficiency, morphology), primary human cells","pmids":["15827126"],"is_preprint":false},{"year":2008,"finding":"Siglec-9 enhances IL-10 production and suppresses TNF-α in macrophages via its cytoplasmic tyrosine-based inhibitory motifs (ITIM). Mutation of both cytoplasmic tyrosines to phenylalanine abolishes the IL-10 enhancement and TNF-α suppression. The membrane-proximal ITIM mutant retains partial TNF-α suppression but loses IL-10 upregulation, indicating distinct regulation of the two cytokines through different ITIM residues.","method":"Stable transfection of RAW264 and THP-1 macrophage lines with wild-type and ITIM-mutant Siglec-9; LPS/CpG/PGN stimulation; ELISA for TNF-α and IL-10","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of ITIM residues with clear mechanistic separation of two cytokine pathways","pmids":["18325328"],"is_preprint":false},{"year":2009,"finding":"Group B Streptococcus (GBS) sialylated capsular polysaccharide (Siaα2-3Galβ1-4GlcNAc) engages neutrophil Siglec-9 via molecular mimicry of host sialoglycans. This interaction dampens neutrophil oxidative burst, reduces NETs formation, and increases bacterial survival. Effects are Sia- and Siglec-9-dependent.","method":"Neutrophil functional assays (oxidative burst, NET formation, bacterial killing) with GBS wild-type and sialic acid-deficient mutants; immobilized synthetic sialoglycan binding; Siglec-9 blocking","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal functional assays with isogenic bacterial mutants and receptor blocking; replicated independently","pmids":["19196661"],"is_preprint":false},{"year":2009,"finding":"Siglec-9 physically interacts with SHP-1 in neutrophils; GM-CSF treatment promotes Siglec-9 phosphorylation in adult PMN but decreases it in neonatal PMN. Neonatal PMN display diminished Siglec-9 expression and constitutive phosphorylation at baseline, associated with altered survival signaling.","method":"Co-immunoprecipitation of Siglec-9 and SHP-1 from neutrophil lysates; immunoblotting; flow cytometry; GM-CSF stimulation experiments","journal":"Pediatric research","confidence":"Medium","confidence_rationale":"Tier 3 — single co-IP/pulldown with partial mechanistic follow-up in primary cells","pmids":["19542910"],"is_preprint":false},{"year":2010,"finding":"MUC16 (CA125) expressed on ovarian cancer cells is identified as the ligand for Siglec-9 on NK cells, B cells, and monocytes. Siglec-9-transfected Jurkat cells and monocytes bind to ovarian tumor cells via Siglec-9–csMUC16 interaction; binding is abolished by neuraminidase treatment, confirming sialic acid dependence.","method":"Siglec-9 transfection of Jurkat cells, neuraminidase treatment, cell adhesion assays, flow cytometry of patient peripheral blood and peritoneal fluid immune cells","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 — Siglec-9 transfection rescue, neuraminidase abolition, adhesion assays with multiple cell types","pmids":["20497550"],"is_preprint":false},{"year":2011,"finding":"Siglec-9 is a leukocyte counter-receptor for vascular adhesion protein-1 (VAP-1/AOC3) on endothelium. The interaction was identified by phage display, confirmed by in vitro and ex vivo adhesion assays, and the binding site was mapped to the enzymatic groove of VAP-1 by molecular modeling and mutant protein assays. Binding is only partially dependent on VAP-1 enzymatic activity. A 68Ga-labeled Siglec-9 peptide specifically detects VAP-1 at inflammatory sites in PET imaging.","method":"Phage display, in vitro and ex vivo adhesion assays, binding assays with mutated VAP-1 proteins, molecular modeling, PET imaging","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 — phage display discovery confirmed by multiple binding assays including mutant proteins and in vivo PET imaging","pmids":["21821708"],"is_preprint":false},{"year":2013,"finding":"Siglec-9 binding to sialylated MUC1 on cancer cells recruits β-catenin to the MUC1 C-terminal domain in a dose- and time-dependent manner, and the recruited β-catenin translocates to the nucleus to promote cell growth. Neuraminidase treatment abolishes the effect, confirming sialic acid dependence.","method":"Co-culture of Siglec-9-expressing HEK293 cells with MUC1-expressing 3T3 and HCT116 cells; β-catenin co-immunoprecipitation and nuclear fractionation; neuraminidase treatment; recombinant soluble Siglec-9 stimulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, neuraminidase control, dose-response, cell co-culture model, nuclear translocation readout","pmids":["24045940"],"is_preprint":false},{"year":2013,"finding":"Siglec-9 binding to sialoglycans on astrocytoma cells causes rapid calpain-mediated degradation of focal adhesion kinase (FAK), Akt, paxillin, and p130Cas, leading to cell detachment, increased motility, and invasiveness. Calpain inhibitors block these effects.","method":"Co-culture of Siglec-9-expressing cells with AS astrocytoma cells; immunoblotting for FAK, Akt, paxillin, p130Cas; calpain inhibitor experiments; motility and invasion assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological inhibition of calpain with multiple substrate readouts, single lab","pmids":["24145038"],"is_preprint":false},{"year":2013,"finding":"Prohibitin-1 and -2 on the surface of T cell lines and activated T lymphocytes act as counter-receptors for Siglec-9 on macrophages/DCs, binding in a sialic acid-independent manner. Mutation of Arg120 to Ala in Siglec-9 abolishes binding, suggesting ionic peptide-peptide interaction. Siglec-9 engagement of prohibitins inhibits ERK1/2 and c-Raf phosphorylation and reduces IL-2 production in Jurkat cells.","method":"Co-immunoprecipitation, bead-based TCR co-stimulation assay with Siglec-9, site-directed mutagenesis (Arg120Ala), immunoblotting for ERK1/2 and c-Raf, IL-2 ELISA","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis plus co-IP and functional signaling readouts, single lab","pmids":["23567969"],"is_preprint":false},{"year":2014,"finding":"Siglec-9 localizes partially to lipid raft (detergent-insoluble microdomain) fractions, and this localization is lectin (sialic acid-binding) dependent. Following TLR2 stimulation, the amount of Siglec-9 in lipid rafts rapidly increases within 3–10 minutes, coinciding with TLR2 recruitment. Lectin-defective Siglec-9 does not enter lipid rafts, and disruption of lipid rafts partially reduces IL-10 production.","method":"Membrane fractionation, detergent-insoluble microdomain isolation, immunoblotting, lectin-defective Siglec-9 mutant, cholesterol oxidase disruption, TLR2 stimulation","journal":"Cytotechnology","confidence":"Medium","confidence_rationale":"Tier 2 — fractionation with functional consequence, lectin-dead mutant control, pharmacological disruption","pmids":["24449467"],"is_preprint":false},{"year":2015,"finding":"Siglec-9 specifically binds high molecular weight hyaluronan (HMW-HA) through a region of the V-set Ig-like domain distinct from the canonical sialic acid-binding site, dampening neutrophil NET formation, oxidative burst, and apoptosis. Group A Streptococcus exploits its HMW-HA capsule to engage this same Siglec-9 binding site, blocking neutrophil killing.","method":"HMW-HA binding assays, neutrophil functional assays (NET formation, oxidative burst, apoptosis), GAS HMW-HA capsule competition experiments, Siglec-9 blocking antibodies","journal":"Journal of molecular medicine","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal functional assays with pathogen mutants and receptor blocking, novel non-sialic acid binding site identified","pmids":["26411873"],"is_preprint":false},{"year":2015,"finding":"Siglec-9 modulates IL-4 responses in macrophages via its cytoplasmic ITIM motifs: Siglec-9 expression enhances arginase-1 (Arg1) induction by IL-4 through MEK/ERK pathway activation. Mutation of cytoplasmic tyrosines in ITIM markedly reduces Arg1 expression. ERK phosphorylation is enhanced basally and MEK inhibitors block the Siglec-9-augmented Arg1 induction, whereas PI-3K inhibitors do not.","method":"Stable transfection of RAW264 with wild-type and ITIM-mutant Siglec-9; IL-4 stimulation; immunoblotting for Akt and ERK phosphorylation; MEK and PI-3K inhibitor treatment; arginase-1 expression assay","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — ITIM mutagenesis with pathway-specific inhibitors and functional readout, single lab","pmids":["26540411"],"is_preprint":false},{"year":2016,"finding":"Cancer-specific MUC1 decorated with short sialylated O-linked glycans (MUC1-ST) engages Siglec-9 on myeloid cells and induces macrophage polarization to a TAM-like phenotype with upregulated PD-L1. Unexpectedly, MUC1-ST/Siglec-9 engagement does not activate SHP-1 or SHP-2 but induces calcium flux leading to MEK-ERK kinase activation.","method":"MUC1-ST–Siglec-9 binding assays, macrophage co-culture, phosphatase activity assays (SHP-1, SHP-2), calcium flux assay, MEK-ERK phosphorylation immunoblotting, PD-L1 flow cytometry","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal signaling assays, unexpected activating pathway characterized rigorously","pmids":["27595232"],"is_preprint":false},{"year":2017,"finding":"Erythrocyte glycophorin A (GYPA), the most abundant sialoglycoprotein on erythrocytes, engages neutrophil Siglec-9 to suppress neutrophil activation (oxidative burst, NET formation, l-selectin shedding, chemotaxis, bacterial killing, and apoptosis). Selective oxidation of sialic acid side chains on erythrocytes reduces Siglec-9 binding and restores neutrophil activation.","method":"Whole blood vs. purified neutrophil comparison, sodium metaperiodate sialic acid oxidation, ELISA and immunofluorescence for GYPA-Siglec-9 engagement, multiple neutrophil functional assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — chemical modification of sialic acids with multiple orthogonal functional readouts; identifies specific ligand GYPA","pmids":["28416510"],"is_preprint":false},{"year":2017,"finding":"Tamm-Horsfall glycoprotein (THP) engages Siglec-9 (and mouse Siglec-E) on neutrophils in a sialic acid (N-glycan)-dependent manner, reducing ROS generation, chemotaxis, and uropathogenic E. coli killing. THP-null mice exhibit significantly more neutrophils in urine, demonstrating a physiological role for THP-Siglec-9 interaction in limiting urinary tract inflammation.","method":"THP-neutrophil binding assays, neuraminidase treatment, neutrophil functional assays, THP-null mouse model, Siglec-E involvement confirmed","journal":"Immunology and cell biology","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout mouse model plus neuraminidase-dependent binding and multiple functional assays","pmids":["28829050"],"is_preprint":false},{"year":2017,"finding":"Soluble Siglec-9 suppresses M1 macrophage activation by inhibiting NF-κB p65 phosphorylation, reduces M1 marker expression (TNF-α, IL-6, iNOS) in RAW264.7 cells, and decreases clinical/histological arthritis severity in collagen-induced arthritis mice, increasing Foxp3+ Treg cells and decreasing serum TNF-α.","method":"RAW264.7 macrophage stimulation assays, NF-κB pathway immunoblotting, collagen-induced arthritis mouse model, in vivo biofluorescence imaging, histology","journal":"Arthritis research & therapy","confidence":"Medium","confidence_rationale":"Tier 2 — NF-κB pathway mechanistic link demonstrated in vitro plus in vivo CIA model, single lab","pmids":["27267914"],"is_preprint":false},{"year":2019,"finding":"Siglec-9+ CD8+ T cells in melanoma tumors are functionally inhibited by Siglec-9 engagement: ligation with Siglec-9 ligands or specific antibodies suppresses TCR signaling, cytotoxicity, and cytokine production, associated with phosphorylation of SHP-1 but not SHP-2.","method":"Flow cytometry of intratumoral vs. peripheral CD8+ T cells, Siglec-9 ligand functional assays, agonist antibody stimulation, SHP-1/SHP-2 phosphorylation immunoblotting, cytotoxicity and cytokine assays","journal":"Cancer immunology research","confidence":"High","confidence_rationale":"Tier 2 — functional inhibition demonstrated with ligands and antibodies, signaling pathway identified with phosphatase specificity","pmids":["30988027"],"is_preprint":false},{"year":2021,"finding":"Pancreatic ductal adenocarcinoma (PDAC) sialic acids synthesized by ST3GAL1 and ST3GAL4 sialyltransferases are recognized by Siglec-9 on myeloid cells, driving monocyte-to-TAM differentiation. Siglec-9 triggering in macrophages reduces inflammatory programs and increases PD-L1 and IL-10 expression.","method":"siRNA knockdown of ST3GAL1/ST3GAL4, Siglec-9 binding assays with PDAC cells, monocyte differentiation assays, PD-L1 and IL-10 expression, single-cell and bulk transcriptomics","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — sialyltransferase knockdown mechanistically links glycan synthesis to Siglec-9 signaling with multiple functional readouts","pmids":["33627655"],"is_preprint":false},{"year":2021,"finding":"Synthetic glycopolymers that act as Siglec-9 agonists suppress NETosis in neutrophils induced by viral TLR agonists and plasma from COVID-19 patients, demonstrating that pharmacological Siglec-9 activation is sufficient to inhibit neutrophil hyperactivation.","method":"Synthetic glycopolymer-mediated Siglec-9 agonism, NETosis assays with TLR agonists and COVID-19 patient plasma","journal":"ACS central science","confidence":"Medium","confidence_rationale":"Tier 2 — direct Siglec-9 agonism with synthetic tool compounds and clear functional readout, single lab","pmids":["34056095"],"is_preprint":false},{"year":2023,"finding":"Siglec-9 acts as an immune checkpoint on macrophages in glioblastoma: Siglec-9 (murine homolog Siglece) deletion activates CD4+ and CD8+ T cells through enhanced antigen presentation, secreted chemokines, and co-stimulatory factor interactions, and synergizes with anti-PD-1/PD-L1 therapy to suppress tumor growth.","method":"Single-cell RNA sequencing, spatial transcriptomics, Siglece knockout mouse models, tumor growth assays, T cell activation assays","journal":"Nature cancer","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout with mechanistic pathway identification and in vivo tumor models","pmids":["37460871"],"is_preprint":false},{"year":2023,"finding":"Siglec-9 is an inhibitory receptor on human mast cells: CRISPR/Cas9 disruption of SIGLEC9 results in increased baseline activation markers and increased responsiveness to IgE-dependent and -independent stimulation. Co-engagement of Siglec-9 with FcεRI reduces degranulation, arachidonic acid production, and chemokine release. Glycophorin A and HMW-HA act as Siglec-9 ligands on mast cells.","method":"CRISPR/Cas9 SIGLEC9 knockout in human mast cells, FcεRI co-engagement assays, degranulation assays, arachidonic acid and cytokine measurement, flow cytometry","journal":"The Journal of allergy and clinical immunology","confidence":"High","confidence_rationale":"Tier 1–2 — CRISPR knockout with gain-of-function phenotype and co-engagement mechanistic assays, multiple readouts","pmids":["37100120"],"is_preprint":false},{"year":2023,"finding":"Blockade of Siglec-9 on TAMs in ovarian cancer suppresses SHP-1 phosphorylation, repolarizes TAMs to an antitumorigenic phenotype, and restores cytotoxic CD8+ T cell activity in vitro and ex vivo.","method":"Flow cytometry, anti-Siglec-9 blocking antibody treatment, SHP-1 phosphorylation assay, macrophage repolarization assays, CD8+ T cell cytotoxicity assays","journal":"Journal for immunotherapy of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic signaling link (SHP-1) with macrophage and T cell functional readouts, single lab","pmids":["37709296"],"is_preprint":false},{"year":2024,"finding":"NMR spectroscopy and molecular dynamics simulations revealed that Neu5Ac is accommodated at the canonical sialic acid-binding site between the F and G β-strands of Siglec-9's V-set domain. Modified sialoglycans with a heteroaromatic scaffold at C9 of Neu5Ac create new interactions with hydrophobic residues at the G-G' loop and N-terminal region; additions at C5 of Neu5Ac stabilize the flexible B'-C loop, explaining enhanced affinity.","method":"Solution NMR spectroscopy (triple resonance 3D NMR backbone assignment), molecular dynamics simulation, binding assays with natural and synthetic sialoglycans","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structure determination combined with MD simulation and binding assays with structure-activity rationale","pmids":["38321945"],"is_preprint":false},{"year":2024,"finding":"ST3GAL4 sialyltransferase is the primary driver of Siglec-9 ligand (α2,3-sialylated N-linked glycan) synthesis in AML cells. CRISPR-Cas9 knockout of ST3GAL4 dramatically reduces Siglec-9 ligand expression and enhances phagocytosis of AML cells by Siglec-9-expressing macrophages.","method":"CRISPR genomic screening, ST3GAL4 CRISPR-Cas9 KO, mass spectrometry glycan analysis, Siglec-9 binding assays, macrophage phagocytosis assays","journal":"Leukemia","confidence":"High","confidence_rationale":"Tier 1–2 — CRISPR KO with glycoproteomic validation and functional phagocytosis assay","pmids":["39551873"],"is_preprint":false},{"year":2024,"finding":"Omicron SARS-CoV-2 RBD binds Siglec-9 on macrophages via the FAPFFAF sequence (positions 371-377); a phenylalanine-to-serine mutation at F375 (F375S) abrogates Siglec-9 binding, restores macrophage phagocytosis and antigen presentation, and enhances immunogenicity of Omicron vaccines.","method":"Reverse mutagenesis of spike protein, Siglec-9 binding assays, macrophage phagocytosis assays, antigen presentation assays, mouse/rabbit/macaque vaccine immunogenicity","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis identifying precise binding motif, multiple functional assays and in vivo vaccine studies","pmids":["38454157"],"is_preprint":false},{"year":2024,"finding":"Sialylated CD59 was identified as a candidate Siglec-9 ligand in prostate cancer using a CRISPRi screen combined with mass spectrometry. Blocking Siglec-7/9–sialic acid interactions inhibited prostate cancer xenograft growth in humanized mice.","method":"CRISPRi screen, mass spectrometry, Siglec-9 ligand binding assays, xenograft mouse model with humanized immune system","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR screen plus MS identifies specific ligand, in vivo validation, single lab","pmids":["39436703"],"is_preprint":false},{"year":2025,"finding":"DSG2 (Desmoglein 2) is identified as a dominant counter-receptor of Siglec-9 in melanoma cells via proximity labeling combined with CRISPR KO screening. The interaction is primarily dependent on sialic acid-bearing N-glycans on DSG2, and blocking DSG2–Siglec-9 significantly enhances macrophage phagocytosis of melanoma cells.","method":"Proximity labeling, CRISPR KO screening, Siglec-9 binding assays, N-glycan dependency assays, macrophage phagocytosis assays","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — proximity labeling discovery confirmed by CRISPR KO and functional phagocytosis assays, single lab","pmids":["39813162"],"is_preprint":false},{"year":2025,"finding":"Siglec-9 suppresses platelet activation through cis-binding to the mucin-like region of GPIbα carrying O-linked α2,3-sialylated glycans on the platelet surface. Conditional knockout of Siglec-E in platelets (platelet factor 4-cre:Siglec-Eflox/flox) increases platelet coagulation activities in vitro and in vivo. The cis-binding GPIbα–Siglec-9 interaction acts as a 'parking brake' on platelet activation.","method":"Conditional Siglec-E knockout mouse model, human platelet in vitro culture, recombinant GPIbα glycoprotein binding assays, neuraminidase treatment, platelet coagulation assays","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 2 — conditional KO mouse model plus human platelet in vitro system and specific ligand identification with neuraminidase control","pmids":["40204021"],"is_preprint":false},{"year":2006,"finding":"Siglec-9 mediates rapid endocytosis of anti-Siglec-9 antibody in AML cells and rat basophilic leukemia cells transfected with Siglec-9, identifying it as an endocytic receptor absent from normal bone marrow myeloid progenitors.","method":"Anti-Siglec-9 mAb endocytosis assays in primary AML cells and transfected RBL cells, flow cytometry","journal":"Leukemia research","confidence":"Medium","confidence_rationale":"Tier 2 — direct endocytosis assay in primary and transfected cells, single lab","pmids":["16828866"],"is_preprint":false}],"current_model":"Siglec-9 is an inhibitory transmembrane lectin receptor that recognizes α2,3- and α2,6-sialylated glycans (and HMW-hyaluronan) via its V-set Ig domain, engaging multiple ligands (MUC1-ST, MUC16, glycophorin A, GPIbα, DSG2, THP, CD59, GPNMB) on immune and tumor cells; upon ligand binding, its cytoplasmic ITIM tyrosines are phosphorylated and recruit SHP-1, suppressing downstream TCR/NK/neutrophil/mast cell activation cascades (ZAP-70, ERK, NFAT), while in specific contexts (MUC1-ST) it paradoxically activates MEK-ERK and calcium flux, and on macrophages it promotes IL-10 production and M2 polarization via NF-κB suppression; additionally, Siglec-9 serves as a ligand for VAP-1/AOC3 on endothelium, mediating leukocyte trafficking to inflammatory sites."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing that Siglec-9 is a sialic acid-binding lectin with defined domain architecture and ITIM-bearing cytoplasmic tail resolved its molecular identity and predicted an inhibitory signaling function.","evidence":"cDNA cloning, COS cell expression, erythrocyte rosetting, mutagenesis of the essential sialic acid-binding arginine, flow cytometry expression profiling across leukocyte lineages","pmids":["10801860","10801862"],"confidence":"High","gaps":["Downstream signaling was not yet demonstrated","No endogenous ligands identified beyond erythrocyte sialic acids"]},{"year":2001,"claim":"Domain-swap mutagenesis of the V-set C-C' loop between Siglec-7 and Siglec-9 revealed the structural determinant of their distinct glycan specificities, establishing the molecular basis for ligand selectivity.","evidence":"Chimeric V-set domain mutagenesis with glyco-probe binding assays in CHO cells","pmids":["11741958"],"confidence":"High","gaps":["Full crystal or NMR structure of the V-set domain not yet determined","In vivo relevance of specificity differences unknown"]},{"year":2004,"claim":"Reconstitution in Jurkat T cells demonstrated that Siglec-9 ITIM recruits SHP-1 upon TCR stimulation, suppresses ZAP-70 phosphorylation and NFAT transcriptional activity, directly proving its inhibitory signaling function.","evidence":"Stable transfection in Jurkat cells, SHP-1 co-immunoprecipitation, NFAT-luciferase reporter, R120 mutagenesis","pmids":["15292262"],"confidence":"High","gaps":["Whether both cytoplasmic tyrosines contribute equally was unclear","Endogenous ligands triggering this pathway in vivo not identified"]},{"year":2005,"claim":"Demonstrating that Siglec-9 ligation induces both caspase-dependent apoptosis and a distinct caspase-independent death in cytokine-primed neutrophils revealed cell-context-dependent functional outcomes.","evidence":"Anti-Siglec-9 ligation on primary neutrophils, caspase inhibitors, ROS scavengers, ROS-deficient patient neutrophils","pmids":["15827126"],"confidence":"High","gaps":["Identity of the endogenous neutrophil Siglec-9 ligand triggering these pathways was unknown","Molecular mechanism of the non-apoptotic death pathway unresolved"]},{"year":2008,"claim":"ITIM tyrosine mutagenesis in macrophages separated IL-10 enhancement from TNF-α suppression, showing that the two ITIM motifs differentially regulate anti- versus pro-inflammatory cytokines.","evidence":"Wild-type and ITIM-mutant Siglec-9 in RAW264/THP-1 macrophages, TLR stimulation, cytokine ELISA","pmids":["18325328"],"confidence":"High","gaps":["Which phosphatase or adaptor is recruited by each individual ITIM was not resolved","In vivo macrophage polarization consequences not tested"]},{"year":2009,"claim":"Group B Streptococcus sialylated capsule was shown to engage neutrophil Siglec-9 to suppress oxidative burst and NET formation, establishing molecular mimicry as a pathogen immune evasion strategy via Siglec-9.","evidence":"Isogenic GBS sialic acid-deficient mutants, neutrophil functional assays, Siglec-9 blocking","pmids":["19196661"],"confidence":"High","gaps":["Whether this extends to other sialylated pathogens beyond GBS was not known","Relative contribution of Siglec-9 vs. other siglecs on neutrophils not quantified"]},{"year":2010,"claim":"MUC16 on ovarian cancer cells was identified as a sialic acid-dependent Siglec-9 ligand on immune cells, providing the first tumor-associated glycoprotein counter-receptor for Siglec-9.","evidence":"Siglec-9 transfection of Jurkat cells, neuraminidase treatment, cell adhesion assays","pmids":["20497550"],"confidence":"High","gaps":["Whether MUC16–Siglec-9 interaction drives functional immune suppression in ovarian cancer was not yet shown","Specific glycan structures on MUC16 mediating binding not characterized"]},{"year":2011,"claim":"Identification of Siglec-9 as a leukocyte counter-receptor for endothelial VAP-1/AOC3 expanded its role beyond immune inhibition to leukocyte trafficking, with binding mapped to the VAP-1 enzymatic groove.","evidence":"Phage display, in vitro/ex vivo adhesion assays, VAP-1 mutant proteins, PET imaging with labeled Siglec-9 peptide","pmids":["21821708"],"confidence":"High","gaps":["Whether Siglec-9–VAP-1 interaction is sialic acid-dependent or peptide-mediated was not fully resolved","In vivo genetic evidence for this trafficking axis not provided"]},{"year":2013,"claim":"Discovery that Siglec-9 binding to sialylated MUC1 on cancer cells activates β-catenin nuclear translocation and that prohibitins serve as sialic acid-independent Siglec-9 counter-receptors on T cells broadened the receptor's signaling repertoire beyond simple inhibition.","evidence":"Co-culture systems with β-catenin co-IP and nuclear fractionation (MUC1); co-IP and ERK/c-Raf signaling with R120A mutagenesis (prohibitins)","pmids":["24045940","23567969"],"confidence":"Medium","gaps":["β-catenin activation by Siglec-9 not confirmed in primary tumor models","Prohibitin binding being sialic acid-independent is unusual and the structural basis is unresolved","Single-lab findings for both observations"]},{"year":2014,"claim":"Siglec-9 was shown to translocate to lipid raft microdomains upon TLR2 stimulation in a lectin-dependent manner, linking its spatial membrane organization to IL-10 regulatory output.","evidence":"Membrane fractionation, lectin-defective mutant, cholesterol disruption, TLR2 stimulation in macrophages","pmids":["24449467"],"confidence":"Medium","gaps":["Direct visualization of Siglec-9 in rafts (e.g. super-resolution microscopy) not performed","Whether raft localization is required for SHP-1 recruitment not tested"]},{"year":2015,"claim":"Identification of HMW-hyaluronan as a non-sialic acid ligand binding a distinct site on Siglec-9's V-set domain, exploited by Group A Streptococcus capsule, revealed a second ligand-recognition mode for immune evasion.","evidence":"HMW-HA binding assays, neutrophil functional assays, GAS capsule competition, Siglec-9 blocking antibodies","pmids":["26411873"],"confidence":"High","gaps":["Structural mapping of the HMW-HA binding site not determined at atomic resolution","Physiological role of HMW-HA–Siglec-9 in tissue homeostasis not established"]},{"year":2016,"claim":"MUC1-ST engagement of Siglec-9 on macrophages was shown to paradoxically activate MEK-ERK and calcium signaling without SHP-1/SHP-2 recruitment, driving TAM polarization and PD-L1 upregulation — fundamentally revising the view that Siglec-9 is exclusively inhibitory.","evidence":"MUC1-ST binding assays, calcium flux, phosphatase activity assays, MEK-ERK immunoblotting, PD-L1 flow cytometry","pmids":["27595232"],"confidence":"High","gaps":["How ligand identity switches Siglec-9 from SHP-1-dependent inhibition to MEK-ERK activation is mechanistically unresolved","Whether this activating mode occurs with other cancer glycoforms is unknown"]},{"year":2017,"claim":"Glycophorin A and Tamm-Horsfall protein were identified as physiological Siglec-9 ligands that suppress neutrophil activation in blood and urinary tract, respectively, establishing tissue-specific immune dampening through distinct sialoglycoprotein ligands.","evidence":"Sialic acid oxidation on erythrocytes, THP-null mouse model, neuraminidase controls, multiple neutrophil functional assays","pmids":["28416510","28829050"],"confidence":"High","gaps":["In vivo human genetic validation of GYPA–Siglec-9 axis missing","Whether other urinary sialoglycoproteins contribute beyond THP not addressed"]},{"year":2019,"claim":"Siglec-9+ CD8+ T cells in melanoma were shown to be functionally inhibited through SHP-1 (not SHP-2) phosphorylation, formally establishing Siglec-9 as a T cell immune checkpoint in the tumor microenvironment.","evidence":"Flow cytometry of intratumoral T cells, agonist antibodies, SHP-1/SHP-2 phosphorylation, cytotoxicity assays","pmids":["30988027"],"confidence":"High","gaps":["Clinical impact of Siglec-9 blockade on T cells not tested in patients","Relative importance vs. PD-1/CTLA-4 checkpoints not compared"]},{"year":2021,"claim":"ST3GAL1/ST3GAL4 sialyltransferases were identified as the enzymes generating Siglec-9 ligands on PDAC cells, mechanistically connecting glycan biosynthesis to myeloid immunosuppression, while synthetic Siglec-9 agonist glycopolymers were shown to suppress NETosis, demonstrating pharmacological tractability.","evidence":"siRNA knockdown of ST3GAL1/4 with binding and macrophage differentiation assays; synthetic glycopolymer Siglec-9 agonism with NETosis assays","pmids":["33627655","34056095"],"confidence":"High","gaps":["Whether blocking sialyltransferases therapeutically is feasible in vivo not shown","Off-target effects of glycopolymer agonists on other siglecs not fully excluded"]},{"year":2023,"claim":"Genetic deletion of Siglec-9 (Siglece in mice) on macrophages in glioblastoma restored antigen presentation and T cell activation, synergizing with anti-PD-1, while CRISPR knockout on human mast cells increased degranulation — demonstrating checkpoint function across multiple immune cell types.","evidence":"Siglece knockout mice with tumor models, scRNA-seq, spatial transcriptomics, CRISPR/Cas9 SIGLEC9 KO in human mast cells, co-engagement assays","pmids":["37460871","37100120"],"confidence":"High","gaps":["Species differences between murine Siglec-E and human Siglec-9 may limit translational inference","Mast cell Siglec-9 signaling pathway details beyond ITIM not characterized"]},{"year":2024,"claim":"Atomic-level understanding of ligand recognition was achieved by NMR, and new ligand axes were identified: ST3GAL4-dependent sialylation drives Siglec-9 engagement on AML cells controlling phagocytosis; Omicron SARS-CoV-2 RBD FAPFFAF motif directly binds Siglec-9 to suppress macrophage function; and CD59 is a Siglec-9 ligand in prostate cancer.","evidence":"NMR/MD simulations of V-set domain; CRISPR screen and glycoproteomics in AML; spike mutagenesis (F375S) with macrophage assays and vaccine immunogenicity; CRISPRi screen in prostate cancer xenografts","pmids":["38321945","39551873","38454157","39436703"],"confidence":"High","gaps":["Full co-crystal structure of Siglec-9 with a glycoprotein ligand not yet solved","Whether SARS-CoV-2 binding is sialic acid-dependent or protein-mediated is not fully delineated","CD59 as Siglec-9 ligand requires independent replication"]},{"year":2025,"claim":"DSG2 was identified as a dominant melanoma ligand for Siglec-9 via proximity labeling, and GPIbα was shown to cis-engage Siglec-9 on platelets to suppress coagulation, extending Siglec-9's inhibitory function to hemostasis.","evidence":"Proximity labeling plus CRISPR KO screening in melanoma; conditional Siglec-E knockout in mouse platelets with human platelet in vitro validation","pmids":["39813162","40204021"],"confidence":"High","gaps":["DSG2 finding from single lab awaiting independent replication","Whether platelet Siglec-9 contributes to bleeding phenotypes in humans is unknown"]},{"year":null,"claim":"Key unresolved questions include how ligand identity switches Siglec-9 between SHP-1-dependent inhibition and MEK-ERK activation, the structural basis for non-sialic acid ligand recognition (HMW-HA, prohibitins, viral RBD), and whether therapeutic Siglec-9 blockade or agonism can be translated to clinical benefit in cancer and inflammatory disease.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No co-crystal structure with a glycoprotein ligand","Mechanism of ligand-dependent signaling mode switching unresolved","Clinical-grade Siglec-9 blocking or agonist agents not yet in trials"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,3,16,20,24]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,5,20,24,31]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,13,31]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,5,6,16,20,21,23,24,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5,15,16,19,20]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[31]}],"complexes":[],"partners":["SHP-1","MUC16","GYPA","AOC3","MUC1","DSG2","GP1BA","CD59"],"other_free_text":[]},"mechanistic_narrative":"Siglec-9 is an inhibitory immunomodulatory receptor expressed on myeloid cells, NK cells, T cell subsets, mast cells, and platelets that broadly dampens innate and adaptive immune activation by recognizing sialylated glycans and recruiting the tyrosine phosphatase SHP-1. Its V-set Ig domain binds α2,3- and α2,6-linked sialic acids on diverse counter-receptors — including MUC16, glycophorin A, Tamm-Horsfall protein, GPIbα, DSG2, CD59, and sialylated bacterial capsules — and also engages high-molecular-weight hyaluronan through a distinct binding site; upon engagement, ITIM-dependent SHP-1 recruitment suppresses TCR signaling (ZAP-70, NFAT), neutrophil oxidative burst and NETosis, mast cell degranulation, and macrophage pro-inflammatory cytokine production while promoting IL-10 secretion and M2/TAM polarization [PMID:15292262, PMID:15827126, PMID:18325328, PMID:37100120, PMID:28416510, PMID:26411873, PMID:33627655]. A ligand-specific exception occurs with cancer-associated sialyl-Tn MUC1, where Siglec-9 instead triggers calcium flux and MEK-ERK activation without SHP-1/SHP-2 engagement, driving PD-L1 upregulation on macrophages [PMID:27595232]. Siglec-9 also functions as a leukocyte counter-receptor for endothelial VAP-1/AOC3, mediating leukocyte trafficking to inflammatory sites [PMID:21821708], and acts as an immune checkpoint in multiple tumor microenvironments where its genetic deletion or antibody blockade restores anti-tumor immunity and synergizes with PD-1/PD-L1 blockade [PMID:37460871, PMID:37709296, PMID:30988027]."},"prefetch_data":{"uniprot":{"accession":"Q9Y336","full_name":"Sialic acid-binding Ig-like lectin 9","aliases":["CDw329","Protein FOAP-9"],"length_aa":463,"mass_kda":50.1,"function":"Putative adhesion molecule that mediates sialic-acid dependent binding to cells. Preferentially binds to alpha-2,3- or alpha-2,6-linked sialic acid. The sialic acid recognition site may be masked by cis interactions with sialic acids on the same cell surface","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y336/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SIGLEC9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SIGLEC9","total_profiled":1310},"omim":[{"mim_id":"606135","title":"KALLIKREIN-RELATED PEPTIDASE 14; KLK14","url":"https://www.omim.org/entry/606135"},{"mim_id":"606094","title":"SIALIC ACID-BINDING IMMUNOGLOBULIN-LIKE LECTIN 12; SIGLEC12","url":"https://www.omim.org/entry/606094"},{"mim_id":"606091","title":"SIALIC ACID-BINDING IMMUNOGLOBULIN-LIKE LECTIN 10; SIGLEC10","url":"https://www.omim.org/entry/606091"},{"mim_id":"605640","title":"SIALIC ACID-BINDING IMMUNOGLOBULIN-LIKE LECTIN 9; SIGLEC9","url":"https://www.omim.org/entry/605640"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":19.9}],"url":"https://www.proteinatlas.org/search/SIGLEC9"},"hgnc":{"alias_symbol":["CD329"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y336","domains":[{"cath_id":"2.60.40.10","chopping":"23-141","consensus_level":"high","plddt":91.3161,"start":23,"end":141},{"cath_id":"2.60.40.10","chopping":"148-234","consensus_level":"high","plddt":86.6776,"start":148,"end":234},{"cath_id":"2.60.40.10","chopping":"241-339","consensus_level":"high","plddt":75.2008,"start":241,"end":339}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y336","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y336-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y336-F1-predicted_aligned_error_v6.png","plddt_mean":73.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SIGLEC9","jax_strain_url":"https://www.jax.org/strain/search?query=SIGLEC9"},"sequence":{"accession":"Q9Y336","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y336.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y336/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y336"}},"corpus_meta":[{"pmid":"19196661","id":"PMC_19196661","title":"Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response.","date":"2009","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/19196661","citation_count":315,"is_preprint":false},{"pmid":"27595232","id":"PMC_27595232","title":"The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9.","date":"2016","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/27595232","citation_count":306,"is_preprint":false},{"pmid":"33627655","id":"PMC_33627655","title":"Sialic acids in pancreatic cancer cells drive tumour-associated macrophage differentiation via the Siglec receptors Siglec-7 and Siglec-9.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33627655","citation_count":243,"is_preprint":false},{"pmid":"10801862","id":"PMC_10801862","title":"Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10801862","citation_count":189,"is_preprint":false},{"pmid":"15827126","id":"PMC_15827126","title":"Siglec-9 transduces apoptotic and nonapoptotic death signals into neutrophils depending on the proinflammatory cytokine environment.","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15827126","citation_count":181,"is_preprint":false},{"pmid":"37460871","id":"PMC_37460871","title":"Siglec-9 acts as an immune-checkpoint molecule on macrophages in glioblastoma, restricting T-cell priming and immunotherapy response.","date":"2023","source":"Nature cancer","url":"https://pubmed.ncbi.nlm.nih.gov/37460871","citation_count":173,"is_preprint":false},{"pmid":"20497550","id":"PMC_20497550","title":"Identification of Siglec-9 as the receptor for MUC16 on human NK cells, B cells, and monocytes.","date":"2010","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/20497550","citation_count":170,"is_preprint":false},{"pmid":"11741958","id":"PMC_11741958","title":"A small region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to alpha 2,8-disialyl and branched alpha 2,6-sialyl residues. 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Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/26411873","citation_count":73,"is_preprint":false},{"pmid":"24045940","id":"PMC_24045940","title":"Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of β-catenin and subsequent cell growth.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24045940","citation_count":66,"is_preprint":false},{"pmid":"20971061","id":"PMC_20971061","title":"Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/20971061","citation_count":66,"is_preprint":false},{"pmid":"25747723","id":"PMC_25747723","title":"Expression of ligands for Siglec-8 and Siglec-9 in human airways and airway cells.","date":"2015","source":"The Journal of allergy and clinical immunology","url":"https://pubmed.ncbi.nlm.nih.gov/25747723","citation_count":52,"is_preprint":false},{"pmid":"34056095","id":"PMC_34056095","title":"Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19.","date":"2021","source":"ACS central science","url":"https://pubmed.ncbi.nlm.nih.gov/34056095","citation_count":52,"is_preprint":false},{"pmid":"16828866","id":"PMC_16828866","title":"Analysis of the CD33-related siglec family reveals that Siglec-9 is an endocytic receptor expressed on subsets of acute myeloid leukemia cells and absent from normal hematopoietic progenitors.","date":"2006","source":"Leukemia research","url":"https://pubmed.ncbi.nlm.nih.gov/16828866","citation_count":51,"is_preprint":false},{"pmid":"32322597","id":"PMC_32322597","title":"The Roles of Siglec7 and Siglec9 on Natural Killer Cells in Virus Infection and Tumour Progression.","date":"2020","source":"Journal of immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/32322597","citation_count":45,"is_preprint":false},{"pmid":"34869028","id":"PMC_34869028","title":"Development of Siglec-9 Blocking Antibody to Enhance Anti-Tumor Immunity.","date":"2021","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/34869028","citation_count":37,"is_preprint":false},{"pmid":"27267914","id":"PMC_27267914","title":"Soluble Siglec-9 suppresses arthritis in a collagen-induced arthritis mouse model and inhibits M1 activation of RAW264.7 macrophages.","date":"2016","source":"Arthritis research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/27267914","citation_count":34,"is_preprint":false},{"pmid":"39436703","id":"PMC_39436703","title":"Sialylated glycoproteins suppress immune cell killing by binding to Siglec-7 and Siglec-9 in prostate cancer.","date":"2024","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/39436703","citation_count":33,"is_preprint":false},{"pmid":"10903842","id":"PMC_10903842","title":"Identification and molecular characterization of a novel member of the siglec family (SIGLEC9).","date":"2000","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10903842","citation_count":33,"is_preprint":false},{"pmid":"29899741","id":"PMC_29899741","title":"Decreased Siglec-9 Expression on Natural Killer Cell Subset Associated With Persistent HBV Replication.","date":"2018","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/29899741","citation_count":32,"is_preprint":false},{"pmid":"28272428","id":"PMC_28272428","title":"Secreted Ectodomain of SIGLEC-9 and MCP-1 Synergistically Improve Acute Liver Failure in Rats by Altering Macrophage Polarity.","date":"2017","source":"Scientific 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journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/28273363","citation_count":15,"is_preprint":false},{"pmid":"36728420","id":"PMC_36728420","title":"Siglec-9 Restrains Antibody-Dependent Natural Killer Cell Cytotoxicity against SARS-CoV-2.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/36728420","citation_count":14,"is_preprint":false},{"pmid":"39551873","id":"PMC_39551873","title":"The glycosyltransferase ST3GAL4 drives immune evasion in acute myeloid leukemia by synthesizing ligands for the glyco-immune checkpoint receptor Siglec-9.","date":"2024","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/39551873","citation_count":14,"is_preprint":false},{"pmid":"26923638","id":"PMC_26923638","title":"Constitutively expressed Siglec-9 inhibits LPS-induced CCR7, but enhances IL-4-induced CD200R expression in human macrophages.","date":"2016","source":"Bioscience, biotechnology, and 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immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38454157","citation_count":12,"is_preprint":false},{"pmid":"24145038","id":"PMC_24145038","title":"Binding of a sialic acid-recognizing lectin Siglec-9 modulates adhesion dynamics of cancer cells via calpain-mediated protein degradation.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24145038","citation_count":12,"is_preprint":false},{"pmid":"23567969","id":"PMC_23567969","title":"Prohibitins function as endogenous ligands for Siglec-9 and negatively regulate TCR signaling upon ligation.","date":"2013","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/23567969","citation_count":10,"is_preprint":false},{"pmid":"36445569","id":"PMC_36445569","title":"Development of Effective Siglec-9 Antibodies Against Cancer.","date":"2022","source":"Current oncology 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/38321945","citation_count":8,"is_preprint":false},{"pmid":"38412768","id":"PMC_38412768","title":"Expression of Siglec-9 in peripheral blood neutrophils was increased and associated with disease severity in patients with AECOPD.","date":"2024","source":"Cytokine","url":"https://pubmed.ncbi.nlm.nih.gov/38412768","citation_count":8,"is_preprint":false},{"pmid":"24882272","id":"PMC_24882272","title":"Dasatinib enhances migration of monocyte-derived dendritic cells by reducing phosphorylation of inhibitory immune receptors Siglec-9 and Siglec-3.","date":"2014","source":"Experimental hematology","url":"https://pubmed.ncbi.nlm.nih.gov/24882272","citation_count":8,"is_preprint":false},{"pmid":"24449467","id":"PMC_24449467","title":"Lectin-dependent localization of cell surface sialic acid-binding lectin Siglec-9.","date":"2014","source":"Cytotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/24449467","citation_count":7,"is_preprint":false},{"pmid":"20733319","id":"PMC_20733319","title":"A novel function of Siglec-9 A391C polymorphism on T cell receptor signaling.","date":"2010","source":"International archives of allergy and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/20733319","citation_count":6,"is_preprint":false},{"pmid":"39813162","id":"PMC_39813162","title":"Proximity Labeling and Genetic Screening Reveal that DSG2 is a Counter Receptor of Siglec-9 and Suppresses Macrophage Phagocytosis.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39813162","citation_count":5,"is_preprint":false},{"pmid":"29391504","id":"PMC_29391504","title":"Mapping the interaction site and effect of the Siglec-9 inflammatory biomarker on human primary amine oxidase.","date":"2018","source":"Scientific 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America","url":"https://pubmed.ncbi.nlm.nih.gov/40892920","citation_count":4,"is_preprint":false},{"pmid":"24464124","id":"PMC_24464124","title":"Enhanced lentiviral vector production in 293FT cells expressing Siglec-9.","date":"2014","source":"Cytotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/24464124","citation_count":4,"is_preprint":false},{"pmid":"37865296","id":"PMC_37865296","title":"The change of Siglec-9 expression in peripheral blood NK cells of SFTS patients can affect the function of NK cells.","date":"2023","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/37865296","citation_count":3,"is_preprint":false},{"pmid":"39727942","id":"PMC_39727942","title":"Association of SIGLEC9 Expression with Cytokine Expression, Tumor Grading, KRAS, NRAS, BRAF, PIK3CA, AKT Gene Mutations, and MSI Status in Colorectal Cancer.","date":"2024","source":"Current issues in molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/39727942","citation_count":3,"is_preprint":false},{"pmid":"39598355","id":"PMC_39598355","title":"Effect of Hypoxia on Siglec-7 and Siglec-9 Receptors and Sialoglycan Ligands and Impact of Their Targeting on NK Cell Cytotoxicity.","date":"2024","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/39598355","citation_count":2,"is_preprint":false},{"pmid":"40204021","id":"PMC_40204021","title":"Sialylated glycoproteins bind to Siglec-9 in a cis manner on platelets to suppress platelet activation.","date":"2025","source":"Journal of thrombosis and haemostasis : JTH","url":"https://pubmed.ncbi.nlm.nih.gov/40204021","citation_count":1,"is_preprint":false},{"pmid":"39037058","id":"PMC_39037058","title":"Transforming the Dark into Light: A Siglec-9 Switch.","date":"2024","source":"Cancer immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/39037058","citation_count":1,"is_preprint":false},{"pmid":"41843671","id":"PMC_41843671","title":"Synthetic SIGLEC9-based chimeric switch receptor augments the efficacy of CAR macrophages against glioblastoma.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41843671","citation_count":0,"is_preprint":false},{"pmid":"40276034","id":"PMC_40276034","title":"Rational design of FVIII sialylated peptides to target Siglec-3 and Siglec-9 and improve peptide formulations for reverse vaccines.","date":"2025","source":"Frontiers in bioengineering and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/40276034","citation_count":0,"is_preprint":false},{"pmid":"40559390","id":"PMC_40559390","title":"Soluble Siglec-9 Improves Intestinal Barrier Function in a Mouse Model of Metabolic Dysfunction-Associated Steatohepatitis.","date":"2025","source":"Metabolites","url":"https://pubmed.ncbi.nlm.nih.gov/40559390","citation_count":0,"is_preprint":false},{"pmid":"41801406","id":"PMC_41801406","title":"Chemoradiotherapy facilitates siglec-10+/siglec-9+ macrophage-mediated impairment of CD24+/MUC16+ tumor cell elimination and enhances PD-L2 dependent immunosuppression in cervical cancer.","date":"2026","source":"Cancer immunology, immunotherapy : CII","url":"https://pubmed.ncbi.nlm.nih.gov/41801406","citation_count":0,"is_preprint":false},{"pmid":"39914835","id":"PMC_39914835","title":"[Preliminary study on the role of siglec-9 expression in peripheral blood of acute respiratory distress syndrome patients].","date":"2025","source":"Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases","url":"https://pubmed.ncbi.nlm.nih.gov/39914835","citation_count":0,"is_preprint":false},{"pmid":"41234517","id":"PMC_41234517","title":"Siglec-9 acts as an immune checkpoint marker on MDSCs in brucella infection.","date":"2025","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/41234517","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.11.09.622776","title":"Integrated multiomics analysis unveils how macrophages drive immune suppression in breast tumors and affect clinical outcomes","date":"2024-11-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.09.622776","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":42153,"output_tokens":8886,"usd":0.129875},"stage2":{"model":"claude-opus-4-6","input_tokens":12762,"output_tokens":4750,"usd":0.27384},"total_usd":0.403715,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"Siglec-9 is a type I transmembrane protein with three extracellular Ig-like domains (N-terminal V-set and two C2-set domains), a transmembrane region, and a cytoplasmic tail containing two tyrosine-based signaling motifs (one ITIM). Expression of full-length cDNA in COS cells induces sialic-acid-dependent erythrocyte binding. Recombinant soluble extracellular domain binds α2-3 and α2-6-linked sialic acids; mutation of a critical arginine in domain 1 abrogates binding.\",\n      \"method\": \"cDNA cloning, COS cell expression, erythrocyte binding assay, recombinant protein binding assay, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in heterologous cells, mutagenesis of active-site arginine, multiple orthogonal binding assays\",\n      \"pmids\": [\"10801860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Siglec-9 is expressed at high or intermediate levels on monocytes, neutrophils, and a minor CD16+/CD56- population; weaker expression on ~50% of B cells and NK cells and minor CD8+ and CD4+ T cell subsets. Binding assays confirmed recognition of sialic acid in α2,3- or α2,6-glycosidic linkage to galactose.\",\n      \"method\": \"Flow cytometry with specific mAb, binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct flow cytometry localization plus binding assays, replicated across multiple labs\",\n      \"pmids\": [\"10801862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-C' loop region (residues Asn70–Lys75) in the V-set sugar-binding domain determines the differential glycan binding specificities of Siglec-7 vs. Siglec-9. Siglec-9 binds LSTc and GD1a oligosaccharides but not GD3 and LSTb, whereas Siglec-7 shows the opposite preference. Substituting this region between the two siglecs swaps their binding specificities.\",\n      \"method\": \"Chimeric V-set domain mutagenesis, polyvalent glyco-probe binding assays on CHO cells, molecular modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain swap mutagenesis combined with structural modeling and glycan binding assays\",\n      \"pmids\": [\"11741958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Siglec-9 negatively regulates T cell receptor (TCR) signaling: upon TCR engagement or pervanadate stimulation, Siglec-9 undergoes tyrosine phosphorylation and recruits SHP-1, reduces phosphorylation of ZAP-70 at Tyr319, and decreases NFAT transcriptional activity. Mutation of the conserved Arg120 in the ligand-binding site reduces inhibitory function, demonstrating that sialic acid ligand binding is required for optimal inhibition.\",\n      \"method\": \"Stable/transient transfection of Jurkat T cells, TCR stimulation assays, SHP-1 co-immunoprecipitation, NFAT/luciferase reporter, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstitution in Jurkat cells, mutagenesis of ligand-binding arginine, multiple downstream readouts\",\n      \"pmids\": [\"15292262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Siglec-9 ligation on neutrophils initiates two death pathways: (1) apoptotic (ROS- and caspase-dependent) under normal conditions, and (2) nonapoptotic/caspase-independent death (characterized by cytoplasmic vacuolization and ROS-dependence) when neutrophils are primed with proinflammatory cytokines (GM-CSF, IFN-α, IFN-γ). ROS scavengers and ROS-deficient neutrophils block both pathways.\",\n      \"method\": \"Siglec-9 ligation on primary neutrophils, caspase inhibitor assays, ROS scavenger experiments, ROS-deficient patient neutrophils, morphological analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (pharmacological inhibition, genetic deficiency, morphology), primary human cells\",\n      \"pmids\": [\"15827126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Siglec-9 enhances IL-10 production and suppresses TNF-α in macrophages via its cytoplasmic tyrosine-based inhibitory motifs (ITIM). Mutation of both cytoplasmic tyrosines to phenylalanine abolishes the IL-10 enhancement and TNF-α suppression. The membrane-proximal ITIM mutant retains partial TNF-α suppression but loses IL-10 upregulation, indicating distinct regulation of the two cytokines through different ITIM residues.\",\n      \"method\": \"Stable transfection of RAW264 and THP-1 macrophage lines with wild-type and ITIM-mutant Siglec-9; LPS/CpG/PGN stimulation; ELISA for TNF-α and IL-10\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of ITIM residues with clear mechanistic separation of two cytokine pathways\",\n      \"pmids\": [\"18325328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Group B Streptococcus (GBS) sialylated capsular polysaccharide (Siaα2-3Galβ1-4GlcNAc) engages neutrophil Siglec-9 via molecular mimicry of host sialoglycans. This interaction dampens neutrophil oxidative burst, reduces NETs formation, and increases bacterial survival. Effects are Sia- and Siglec-9-dependent.\",\n      \"method\": \"Neutrophil functional assays (oxidative burst, NET formation, bacterial killing) with GBS wild-type and sialic acid-deficient mutants; immobilized synthetic sialoglycan binding; Siglec-9 blocking\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal functional assays with isogenic bacterial mutants and receptor blocking; replicated independently\",\n      \"pmids\": [\"19196661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Siglec-9 physically interacts with SHP-1 in neutrophils; GM-CSF treatment promotes Siglec-9 phosphorylation in adult PMN but decreases it in neonatal PMN. Neonatal PMN display diminished Siglec-9 expression and constitutive phosphorylation at baseline, associated with altered survival signaling.\",\n      \"method\": \"Co-immunoprecipitation of Siglec-9 and SHP-1 from neutrophil lysates; immunoblotting; flow cytometry; GM-CSF stimulation experiments\",\n      \"journal\": \"Pediatric research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP/pulldown with partial mechanistic follow-up in primary cells\",\n      \"pmids\": [\"19542910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MUC16 (CA125) expressed on ovarian cancer cells is identified as the ligand for Siglec-9 on NK cells, B cells, and monocytes. Siglec-9-transfected Jurkat cells and monocytes bind to ovarian tumor cells via Siglec-9–csMUC16 interaction; binding is abolished by neuraminidase treatment, confirming sialic acid dependence.\",\n      \"method\": \"Siglec-9 transfection of Jurkat cells, neuraminidase treatment, cell adhesion assays, flow cytometry of patient peripheral blood and peritoneal fluid immune cells\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Siglec-9 transfection rescue, neuraminidase abolition, adhesion assays with multiple cell types\",\n      \"pmids\": [\"20497550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Siglec-9 is a leukocyte counter-receptor for vascular adhesion protein-1 (VAP-1/AOC3) on endothelium. The interaction was identified by phage display, confirmed by in vitro and ex vivo adhesion assays, and the binding site was mapped to the enzymatic groove of VAP-1 by molecular modeling and mutant protein assays. Binding is only partially dependent on VAP-1 enzymatic activity. A 68Ga-labeled Siglec-9 peptide specifically detects VAP-1 at inflammatory sites in PET imaging.\",\n      \"method\": \"Phage display, in vitro and ex vivo adhesion assays, binding assays with mutated VAP-1 proteins, molecular modeling, PET imaging\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — phage display discovery confirmed by multiple binding assays including mutant proteins and in vivo PET imaging\",\n      \"pmids\": [\"21821708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Siglec-9 binding to sialylated MUC1 on cancer cells recruits β-catenin to the MUC1 C-terminal domain in a dose- and time-dependent manner, and the recruited β-catenin translocates to the nucleus to promote cell growth. Neuraminidase treatment abolishes the effect, confirming sialic acid dependence.\",\n      \"method\": \"Co-culture of Siglec-9-expressing HEK293 cells with MUC1-expressing 3T3 and HCT116 cells; β-catenin co-immunoprecipitation and nuclear fractionation; neuraminidase treatment; recombinant soluble Siglec-9 stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, neuraminidase control, dose-response, cell co-culture model, nuclear translocation readout\",\n      \"pmids\": [\"24045940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Siglec-9 binding to sialoglycans on astrocytoma cells causes rapid calpain-mediated degradation of focal adhesion kinase (FAK), Akt, paxillin, and p130Cas, leading to cell detachment, increased motility, and invasiveness. Calpain inhibitors block these effects.\",\n      \"method\": \"Co-culture of Siglec-9-expressing cells with AS astrocytoma cells; immunoblotting for FAK, Akt, paxillin, p130Cas; calpain inhibitor experiments; motility and invasion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological inhibition of calpain with multiple substrate readouts, single lab\",\n      \"pmids\": [\"24145038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Prohibitin-1 and -2 on the surface of T cell lines and activated T lymphocytes act as counter-receptors for Siglec-9 on macrophages/DCs, binding in a sialic acid-independent manner. Mutation of Arg120 to Ala in Siglec-9 abolishes binding, suggesting ionic peptide-peptide interaction. Siglec-9 engagement of prohibitins inhibits ERK1/2 and c-Raf phosphorylation and reduces IL-2 production in Jurkat cells.\",\n      \"method\": \"Co-immunoprecipitation, bead-based TCR co-stimulation assay with Siglec-9, site-directed mutagenesis (Arg120Ala), immunoblotting for ERK1/2 and c-Raf, IL-2 ELISA\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis plus co-IP and functional signaling readouts, single lab\",\n      \"pmids\": [\"23567969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Siglec-9 localizes partially to lipid raft (detergent-insoluble microdomain) fractions, and this localization is lectin (sialic acid-binding) dependent. Following TLR2 stimulation, the amount of Siglec-9 in lipid rafts rapidly increases within 3–10 minutes, coinciding with TLR2 recruitment. Lectin-defective Siglec-9 does not enter lipid rafts, and disruption of lipid rafts partially reduces IL-10 production.\",\n      \"method\": \"Membrane fractionation, detergent-insoluble microdomain isolation, immunoblotting, lectin-defective Siglec-9 mutant, cholesterol oxidase disruption, TLR2 stimulation\",\n      \"journal\": \"Cytotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — fractionation with functional consequence, lectin-dead mutant control, pharmacological disruption\",\n      \"pmids\": [\"24449467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Siglec-9 specifically binds high molecular weight hyaluronan (HMW-HA) through a region of the V-set Ig-like domain distinct from the canonical sialic acid-binding site, dampening neutrophil NET formation, oxidative burst, and apoptosis. Group A Streptococcus exploits its HMW-HA capsule to engage this same Siglec-9 binding site, blocking neutrophil killing.\",\n      \"method\": \"HMW-HA binding assays, neutrophil functional assays (NET formation, oxidative burst, apoptosis), GAS HMW-HA capsule competition experiments, Siglec-9 blocking antibodies\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal functional assays with pathogen mutants and receptor blocking, novel non-sialic acid binding site identified\",\n      \"pmids\": [\"26411873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Siglec-9 modulates IL-4 responses in macrophages via its cytoplasmic ITIM motifs: Siglec-9 expression enhances arginase-1 (Arg1) induction by IL-4 through MEK/ERK pathway activation. Mutation of cytoplasmic tyrosines in ITIM markedly reduces Arg1 expression. ERK phosphorylation is enhanced basally and MEK inhibitors block the Siglec-9-augmented Arg1 induction, whereas PI-3K inhibitors do not.\",\n      \"method\": \"Stable transfection of RAW264 with wild-type and ITIM-mutant Siglec-9; IL-4 stimulation; immunoblotting for Akt and ERK phosphorylation; MEK and PI-3K inhibitor treatment; arginase-1 expression assay\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ITIM mutagenesis with pathway-specific inhibitors and functional readout, single lab\",\n      \"pmids\": [\"26540411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cancer-specific MUC1 decorated with short sialylated O-linked glycans (MUC1-ST) engages Siglec-9 on myeloid cells and induces macrophage polarization to a TAM-like phenotype with upregulated PD-L1. Unexpectedly, MUC1-ST/Siglec-9 engagement does not activate SHP-1 or SHP-2 but induces calcium flux leading to MEK-ERK kinase activation.\",\n      \"method\": \"MUC1-ST–Siglec-9 binding assays, macrophage co-culture, phosphatase activity assays (SHP-1, SHP-2), calcium flux assay, MEK-ERK phosphorylation immunoblotting, PD-L1 flow cytometry\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal signaling assays, unexpected activating pathway characterized rigorously\",\n      \"pmids\": [\"27595232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Erythrocyte glycophorin A (GYPA), the most abundant sialoglycoprotein on erythrocytes, engages neutrophil Siglec-9 to suppress neutrophil activation (oxidative burst, NET formation, l-selectin shedding, chemotaxis, bacterial killing, and apoptosis). Selective oxidation of sialic acid side chains on erythrocytes reduces Siglec-9 binding and restores neutrophil activation.\",\n      \"method\": \"Whole blood vs. purified neutrophil comparison, sodium metaperiodate sialic acid oxidation, ELISA and immunofluorescence for GYPA-Siglec-9 engagement, multiple neutrophil functional assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chemical modification of sialic acids with multiple orthogonal functional readouts; identifies specific ligand GYPA\",\n      \"pmids\": [\"28416510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tamm-Horsfall glycoprotein (THP) engages Siglec-9 (and mouse Siglec-E) on neutrophils in a sialic acid (N-glycan)-dependent manner, reducing ROS generation, chemotaxis, and uropathogenic E. coli killing. THP-null mice exhibit significantly more neutrophils in urine, demonstrating a physiological role for THP-Siglec-9 interaction in limiting urinary tract inflammation.\",\n      \"method\": \"THP-neutrophil binding assays, neuraminidase treatment, neutrophil functional assays, THP-null mouse model, Siglec-E involvement confirmed\",\n      \"journal\": \"Immunology and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout mouse model plus neuraminidase-dependent binding and multiple functional assays\",\n      \"pmids\": [\"28829050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Soluble Siglec-9 suppresses M1 macrophage activation by inhibiting NF-κB p65 phosphorylation, reduces M1 marker expression (TNF-α, IL-6, iNOS) in RAW264.7 cells, and decreases clinical/histological arthritis severity in collagen-induced arthritis mice, increasing Foxp3+ Treg cells and decreasing serum TNF-α.\",\n      \"method\": \"RAW264.7 macrophage stimulation assays, NF-κB pathway immunoblotting, collagen-induced arthritis mouse model, in vivo biofluorescence imaging, histology\",\n      \"journal\": \"Arthritis research & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — NF-κB pathway mechanistic link demonstrated in vitro plus in vivo CIA model, single lab\",\n      \"pmids\": [\"27267914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Siglec-9+ CD8+ T cells in melanoma tumors are functionally inhibited by Siglec-9 engagement: ligation with Siglec-9 ligands or specific antibodies suppresses TCR signaling, cytotoxicity, and cytokine production, associated with phosphorylation of SHP-1 but not SHP-2.\",\n      \"method\": \"Flow cytometry of intratumoral vs. peripheral CD8+ T cells, Siglec-9 ligand functional assays, agonist antibody stimulation, SHP-1/SHP-2 phosphorylation immunoblotting, cytotoxicity and cytokine assays\",\n      \"journal\": \"Cancer immunology research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional inhibition demonstrated with ligands and antibodies, signaling pathway identified with phosphatase specificity\",\n      \"pmids\": [\"30988027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pancreatic ductal adenocarcinoma (PDAC) sialic acids synthesized by ST3GAL1 and ST3GAL4 sialyltransferases are recognized by Siglec-9 on myeloid cells, driving monocyte-to-TAM differentiation. Siglec-9 triggering in macrophages reduces inflammatory programs and increases PD-L1 and IL-10 expression.\",\n      \"method\": \"siRNA knockdown of ST3GAL1/ST3GAL4, Siglec-9 binding assays with PDAC cells, monocyte differentiation assays, PD-L1 and IL-10 expression, single-cell and bulk transcriptomics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — sialyltransferase knockdown mechanistically links glycan synthesis to Siglec-9 signaling with multiple functional readouts\",\n      \"pmids\": [\"33627655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Synthetic glycopolymers that act as Siglec-9 agonists suppress NETosis in neutrophils induced by viral TLR agonists and plasma from COVID-19 patients, demonstrating that pharmacological Siglec-9 activation is sufficient to inhibit neutrophil hyperactivation.\",\n      \"method\": \"Synthetic glycopolymer-mediated Siglec-9 agonism, NETosis assays with TLR agonists and COVID-19 patient plasma\",\n      \"journal\": \"ACS central science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct Siglec-9 agonism with synthetic tool compounds and clear functional readout, single lab\",\n      \"pmids\": [\"34056095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Siglec-9 acts as an immune checkpoint on macrophages in glioblastoma: Siglec-9 (murine homolog Siglece) deletion activates CD4+ and CD8+ T cells through enhanced antigen presentation, secreted chemokines, and co-stimulatory factor interactions, and synergizes with anti-PD-1/PD-L1 therapy to suppress tumor growth.\",\n      \"method\": \"Single-cell RNA sequencing, spatial transcriptomics, Siglece knockout mouse models, tumor growth assays, T cell activation assays\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout with mechanistic pathway identification and in vivo tumor models\",\n      \"pmids\": [\"37460871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Siglec-9 is an inhibitory receptor on human mast cells: CRISPR/Cas9 disruption of SIGLEC9 results in increased baseline activation markers and increased responsiveness to IgE-dependent and -independent stimulation. Co-engagement of Siglec-9 with FcεRI reduces degranulation, arachidonic acid production, and chemokine release. Glycophorin A and HMW-HA act as Siglec-9 ligands on mast cells.\",\n      \"method\": \"CRISPR/Cas9 SIGLEC9 knockout in human mast cells, FcεRI co-engagement assays, degranulation assays, arachidonic acid and cytokine measurement, flow cytometry\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — CRISPR knockout with gain-of-function phenotype and co-engagement mechanistic assays, multiple readouts\",\n      \"pmids\": [\"37100120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Blockade of Siglec-9 on TAMs in ovarian cancer suppresses SHP-1 phosphorylation, repolarizes TAMs to an antitumorigenic phenotype, and restores cytotoxic CD8+ T cell activity in vitro and ex vivo.\",\n      \"method\": \"Flow cytometry, anti-Siglec-9 blocking antibody treatment, SHP-1 phosphorylation assay, macrophage repolarization assays, CD8+ T cell cytotoxicity assays\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic signaling link (SHP-1) with macrophage and T cell functional readouts, single lab\",\n      \"pmids\": [\"37709296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NMR spectroscopy and molecular dynamics simulations revealed that Neu5Ac is accommodated at the canonical sialic acid-binding site between the F and G β-strands of Siglec-9's V-set domain. Modified sialoglycans with a heteroaromatic scaffold at C9 of Neu5Ac create new interactions with hydrophobic residues at the G-G' loop and N-terminal region; additions at C5 of Neu5Ac stabilize the flexible B'-C loop, explaining enhanced affinity.\",\n      \"method\": \"Solution NMR spectroscopy (triple resonance 3D NMR backbone assignment), molecular dynamics simulation, binding assays with natural and synthetic sialoglycans\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure determination combined with MD simulation and binding assays with structure-activity rationale\",\n      \"pmids\": [\"38321945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ST3GAL4 sialyltransferase is the primary driver of Siglec-9 ligand (α2,3-sialylated N-linked glycan) synthesis in AML cells. CRISPR-Cas9 knockout of ST3GAL4 dramatically reduces Siglec-9 ligand expression and enhances phagocytosis of AML cells by Siglec-9-expressing macrophages.\",\n      \"method\": \"CRISPR genomic screening, ST3GAL4 CRISPR-Cas9 KO, mass spectrometry glycan analysis, Siglec-9 binding assays, macrophage phagocytosis assays\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — CRISPR KO with glycoproteomic validation and functional phagocytosis assay\",\n      \"pmids\": [\"39551873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Omicron SARS-CoV-2 RBD binds Siglec-9 on macrophages via the FAPFFAF sequence (positions 371-377); a phenylalanine-to-serine mutation at F375 (F375S) abrogates Siglec-9 binding, restores macrophage phagocytosis and antigen presentation, and enhances immunogenicity of Omicron vaccines.\",\n      \"method\": \"Reverse mutagenesis of spike protein, Siglec-9 binding assays, macrophage phagocytosis assays, antigen presentation assays, mouse/rabbit/macaque vaccine immunogenicity\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis identifying precise binding motif, multiple functional assays and in vivo vaccine studies\",\n      \"pmids\": [\"38454157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Sialylated CD59 was identified as a candidate Siglec-9 ligand in prostate cancer using a CRISPRi screen combined with mass spectrometry. Blocking Siglec-7/9–sialic acid interactions inhibited prostate cancer xenograft growth in humanized mice.\",\n      \"method\": \"CRISPRi screen, mass spectrometry, Siglec-9 ligand binding assays, xenograft mouse model with humanized immune system\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR screen plus MS identifies specific ligand, in vivo validation, single lab\",\n      \"pmids\": [\"39436703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DSG2 (Desmoglein 2) is identified as a dominant counter-receptor of Siglec-9 in melanoma cells via proximity labeling combined with CRISPR KO screening. The interaction is primarily dependent on sialic acid-bearing N-glycans on DSG2, and blocking DSG2–Siglec-9 significantly enhances macrophage phagocytosis of melanoma cells.\",\n      \"method\": \"Proximity labeling, CRISPR KO screening, Siglec-9 binding assays, N-glycan dependency assays, macrophage phagocytosis assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proximity labeling discovery confirmed by CRISPR KO and functional phagocytosis assays, single lab\",\n      \"pmids\": [\"39813162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Siglec-9 suppresses platelet activation through cis-binding to the mucin-like region of GPIbα carrying O-linked α2,3-sialylated glycans on the platelet surface. Conditional knockout of Siglec-E in platelets (platelet factor 4-cre:Siglec-Eflox/flox) increases platelet coagulation activities in vitro and in vivo. The cis-binding GPIbα–Siglec-9 interaction acts as a 'parking brake' on platelet activation.\",\n      \"method\": \"Conditional Siglec-E knockout mouse model, human platelet in vitro culture, recombinant GPIbα glycoprotein binding assays, neuraminidase treatment, platelet coagulation assays\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO mouse model plus human platelet in vitro system and specific ligand identification with neuraminidase control\",\n      \"pmids\": [\"40204021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Siglec-9 mediates rapid endocytosis of anti-Siglec-9 antibody in AML cells and rat basophilic leukemia cells transfected with Siglec-9, identifying it as an endocytic receptor absent from normal bone marrow myeloid progenitors.\",\n      \"method\": \"Anti-Siglec-9 mAb endocytosis assays in primary AML cells and transfected RBL cells, flow cytometry\",\n      \"journal\": \"Leukemia research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct endocytosis assay in primary and transfected cells, single lab\",\n      \"pmids\": [\"16828866\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Siglec-9 is an inhibitory transmembrane lectin receptor that recognizes α2,3- and α2,6-sialylated glycans (and HMW-hyaluronan) via its V-set Ig domain, engaging multiple ligands (MUC1-ST, MUC16, glycophorin A, GPIbα, DSG2, THP, CD59, GPNMB) on immune and tumor cells; upon ligand binding, its cytoplasmic ITIM tyrosines are phosphorylated and recruit SHP-1, suppressing downstream TCR/NK/neutrophil/mast cell activation cascades (ZAP-70, ERK, NFAT), while in specific contexts (MUC1-ST) it paradoxically activates MEK-ERK and calcium flux, and on macrophages it promotes IL-10 production and M2 polarization via NF-κB suppression; additionally, Siglec-9 serves as a ligand for VAP-1/AOC3 on endothelium, mediating leukocyte trafficking to inflammatory sites.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Siglec-9 is an inhibitory immunomodulatory receptor expressed on myeloid cells, NK cells, T cell subsets, mast cells, and platelets that broadly dampens innate and adaptive immune activation by recognizing sialylated glycans and recruiting the tyrosine phosphatase SHP-1. Its V-set Ig domain binds α2,3- and α2,6-linked sialic acids on diverse counter-receptors — including MUC16, glycophorin A, Tamm-Horsfall protein, GPIbα, DSG2, CD59, and sialylated bacterial capsules — and also engages high-molecular-weight hyaluronan through a distinct binding site; upon engagement, ITIM-dependent SHP-1 recruitment suppresses TCR signaling (ZAP-70, NFAT), neutrophil oxidative burst and NETosis, mast cell degranulation, and macrophage pro-inflammatory cytokine production while promoting IL-10 secretion and M2/TAM polarization [PMID:15292262, PMID:15827126, PMID:18325328, PMID:37100120, PMID:28416510, PMID:26411873, PMID:33627655]. A ligand-specific exception occurs with cancer-associated sialyl-Tn MUC1, where Siglec-9 instead triggers calcium flux and MEK-ERK activation without SHP-1/SHP-2 engagement, driving PD-L1 upregulation on macrophages [PMID:27595232]. Siglec-9 also functions as a leukocyte counter-receptor for endothelial VAP-1/AOC3, mediating leukocyte trafficking to inflammatory sites [PMID:21821708], and acts as an immune checkpoint in multiple tumor microenvironments where its genetic deletion or antibody blockade restores anti-tumor immunity and synergizes with PD-1/PD-L1 blockade [PMID:37460871, PMID:37709296, PMID:30988027].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing that Siglec-9 is a sialic acid-binding lectin with defined domain architecture and ITIM-bearing cytoplasmic tail resolved its molecular identity and predicted an inhibitory signaling function.\",\n      \"evidence\": \"cDNA cloning, COS cell expression, erythrocyte rosetting, mutagenesis of the essential sialic acid-binding arginine, flow cytometry expression profiling across leukocyte lineages\",\n      \"pmids\": [\"10801860\", \"10801862\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling was not yet demonstrated\", \"No endogenous ligands identified beyond erythrocyte sialic acids\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Domain-swap mutagenesis of the V-set C-C' loop between Siglec-7 and Siglec-9 revealed the structural determinant of their distinct glycan specificities, establishing the molecular basis for ligand selectivity.\",\n      \"evidence\": \"Chimeric V-set domain mutagenesis with glyco-probe binding assays in CHO cells\",\n      \"pmids\": [\"11741958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full crystal or NMR structure of the V-set domain not yet determined\", \"In vivo relevance of specificity differences unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Reconstitution in Jurkat T cells demonstrated that Siglec-9 ITIM recruits SHP-1 upon TCR stimulation, suppresses ZAP-70 phosphorylation and NFAT transcriptional activity, directly proving its inhibitory signaling function.\",\n      \"evidence\": \"Stable transfection in Jurkat cells, SHP-1 co-immunoprecipitation, NFAT-luciferase reporter, R120 mutagenesis\",\n      \"pmids\": [\"15292262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether both cytoplasmic tyrosines contribute equally was unclear\", \"Endogenous ligands triggering this pathway in vivo not identified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrating that Siglec-9 ligation induces both caspase-dependent apoptosis and a distinct caspase-independent death in cytokine-primed neutrophils revealed cell-context-dependent functional outcomes.\",\n      \"evidence\": \"Anti-Siglec-9 ligation on primary neutrophils, caspase inhibitors, ROS scavengers, ROS-deficient patient neutrophils\",\n      \"pmids\": [\"15827126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the endogenous neutrophil Siglec-9 ligand triggering these pathways was unknown\", \"Molecular mechanism of the non-apoptotic death pathway unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"ITIM tyrosine mutagenesis in macrophages separated IL-10 enhancement from TNF-α suppression, showing that the two ITIM motifs differentially regulate anti- versus pro-inflammatory cytokines.\",\n      \"evidence\": \"Wild-type and ITIM-mutant Siglec-9 in RAW264/THP-1 macrophages, TLR stimulation, cytokine ELISA\",\n      \"pmids\": [\"18325328\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which phosphatase or adaptor is recruited by each individual ITIM was not resolved\", \"In vivo macrophage polarization consequences not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Group B Streptococcus sialylated capsule was shown to engage neutrophil Siglec-9 to suppress oxidative burst and NET formation, establishing molecular mimicry as a pathogen immune evasion strategy via Siglec-9.\",\n      \"evidence\": \"Isogenic GBS sialic acid-deficient mutants, neutrophil functional assays, Siglec-9 blocking\",\n      \"pmids\": [\"19196661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this extends to other sialylated pathogens beyond GBS was not known\", \"Relative contribution of Siglec-9 vs. other siglecs on neutrophils not quantified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"MUC16 on ovarian cancer cells was identified as a sialic acid-dependent Siglec-9 ligand on immune cells, providing the first tumor-associated glycoprotein counter-receptor for Siglec-9.\",\n      \"evidence\": \"Siglec-9 transfection of Jurkat cells, neuraminidase treatment, cell adhesion assays\",\n      \"pmids\": [\"20497550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MUC16–Siglec-9 interaction drives functional immune suppression in ovarian cancer was not yet shown\", \"Specific glycan structures on MUC16 mediating binding not characterized\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of Siglec-9 as a leukocyte counter-receptor for endothelial VAP-1/AOC3 expanded its role beyond immune inhibition to leukocyte trafficking, with binding mapped to the VAP-1 enzymatic groove.\",\n      \"evidence\": \"Phage display, in vitro/ex vivo adhesion assays, VAP-1 mutant proteins, PET imaging with labeled Siglec-9 peptide\",\n      \"pmids\": [\"21821708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Siglec-9–VAP-1 interaction is sialic acid-dependent or peptide-mediated was not fully resolved\", \"In vivo genetic evidence for this trafficking axis not provided\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovery that Siglec-9 binding to sialylated MUC1 on cancer cells activates β-catenin nuclear translocation and that prohibitins serve as sialic acid-independent Siglec-9 counter-receptors on T cells broadened the receptor's signaling repertoire beyond simple inhibition.\",\n      \"evidence\": \"Co-culture systems with β-catenin co-IP and nuclear fractionation (MUC1); co-IP and ERK/c-Raf signaling with R120A mutagenesis (prohibitins)\",\n      \"pmids\": [\"24045940\", \"23567969\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"β-catenin activation by Siglec-9 not confirmed in primary tumor models\", \"Prohibitin binding being sialic acid-independent is unusual and the structural basis is unresolved\", \"Single-lab findings for both observations\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Siglec-9 was shown to translocate to lipid raft microdomains upon TLR2 stimulation in a lectin-dependent manner, linking its spatial membrane organization to IL-10 regulatory output.\",\n      \"evidence\": \"Membrane fractionation, lectin-defective mutant, cholesterol disruption, TLR2 stimulation in macrophages\",\n      \"pmids\": [\"24449467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct visualization of Siglec-9 in rafts (e.g. super-resolution microscopy) not performed\", \"Whether raft localization is required for SHP-1 recruitment not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of HMW-hyaluronan as a non-sialic acid ligand binding a distinct site on Siglec-9's V-set domain, exploited by Group A Streptococcus capsule, revealed a second ligand-recognition mode for immune evasion.\",\n      \"evidence\": \"HMW-HA binding assays, neutrophil functional assays, GAS capsule competition, Siglec-9 blocking antibodies\",\n      \"pmids\": [\"26411873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mapping of the HMW-HA binding site not determined at atomic resolution\", \"Physiological role of HMW-HA–Siglec-9 in tissue homeostasis not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"MUC1-ST engagement of Siglec-9 on macrophages was shown to paradoxically activate MEK-ERK and calcium signaling without SHP-1/SHP-2 recruitment, driving TAM polarization and PD-L1 upregulation — fundamentally revising the view that Siglec-9 is exclusively inhibitory.\",\n      \"evidence\": \"MUC1-ST binding assays, calcium flux, phosphatase activity assays, MEK-ERK immunoblotting, PD-L1 flow cytometry\",\n      \"pmids\": [\"27595232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ligand identity switches Siglec-9 from SHP-1-dependent inhibition to MEK-ERK activation is mechanistically unresolved\", \"Whether this activating mode occurs with other cancer glycoforms is unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Glycophorin A and Tamm-Horsfall protein were identified as physiological Siglec-9 ligands that suppress neutrophil activation in blood and urinary tract, respectively, establishing tissue-specific immune dampening through distinct sialoglycoprotein ligands.\",\n      \"evidence\": \"Sialic acid oxidation on erythrocytes, THP-null mouse model, neuraminidase controls, multiple neutrophil functional assays\",\n      \"pmids\": [\"28416510\", \"28829050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo human genetic validation of GYPA–Siglec-9 axis missing\", \"Whether other urinary sialoglycoproteins contribute beyond THP not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Siglec-9+ CD8+ T cells in melanoma were shown to be functionally inhibited through SHP-1 (not SHP-2) phosphorylation, formally establishing Siglec-9 as a T cell immune checkpoint in the tumor microenvironment.\",\n      \"evidence\": \"Flow cytometry of intratumoral T cells, agonist antibodies, SHP-1/SHP-2 phosphorylation, cytotoxicity assays\",\n      \"pmids\": [\"30988027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Clinical impact of Siglec-9 blockade on T cells not tested in patients\", \"Relative importance vs. PD-1/CTLA-4 checkpoints not compared\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"ST3GAL1/ST3GAL4 sialyltransferases were identified as the enzymes generating Siglec-9 ligands on PDAC cells, mechanistically connecting glycan biosynthesis to myeloid immunosuppression, while synthetic Siglec-9 agonist glycopolymers were shown to suppress NETosis, demonstrating pharmacological tractability.\",\n      \"evidence\": \"siRNA knockdown of ST3GAL1/4 with binding and macrophage differentiation assays; synthetic glycopolymer Siglec-9 agonism with NETosis assays\",\n      \"pmids\": [\"33627655\", \"34056095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether blocking sialyltransferases therapeutically is feasible in vivo not shown\", \"Off-target effects of glycopolymer agonists on other siglecs not fully excluded\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Genetic deletion of Siglec-9 (Siglece in mice) on macrophages in glioblastoma restored antigen presentation and T cell activation, synergizing with anti-PD-1, while CRISPR knockout on human mast cells increased degranulation — demonstrating checkpoint function across multiple immune cell types.\",\n      \"evidence\": \"Siglece knockout mice with tumor models, scRNA-seq, spatial transcriptomics, CRISPR/Cas9 SIGLEC9 KO in human mast cells, co-engagement assays\",\n      \"pmids\": [\"37460871\", \"37100120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Species differences between murine Siglec-E and human Siglec-9 may limit translational inference\", \"Mast cell Siglec-9 signaling pathway details beyond ITIM not characterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Atomic-level understanding of ligand recognition was achieved by NMR, and new ligand axes were identified: ST3GAL4-dependent sialylation drives Siglec-9 engagement on AML cells controlling phagocytosis; Omicron SARS-CoV-2 RBD FAPFFAF motif directly binds Siglec-9 to suppress macrophage function; and CD59 is a Siglec-9 ligand in prostate cancer.\",\n      \"evidence\": \"NMR/MD simulations of V-set domain; CRISPR screen and glycoproteomics in AML; spike mutagenesis (F375S) with macrophage assays and vaccine immunogenicity; CRISPRi screen in prostate cancer xenografts\",\n      \"pmids\": [\"38321945\", \"39551873\", \"38454157\", \"39436703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full co-crystal structure of Siglec-9 with a glycoprotein ligand not yet solved\", \"Whether SARS-CoV-2 binding is sialic acid-dependent or protein-mediated is not fully delineated\", \"CD59 as Siglec-9 ligand requires independent replication\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"DSG2 was identified as a dominant melanoma ligand for Siglec-9 via proximity labeling, and GPIbα was shown to cis-engage Siglec-9 on platelets to suppress coagulation, extending Siglec-9's inhibitory function to hemostasis.\",\n      \"evidence\": \"Proximity labeling plus CRISPR KO screening in melanoma; conditional Siglec-E knockout in mouse platelets with human platelet in vitro validation\",\n      \"pmids\": [\"39813162\", \"40204021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DSG2 finding from single lab awaiting independent replication\", \"Whether platelet Siglec-9 contributes to bleeding phenotypes in humans is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how ligand identity switches Siglec-9 between SHP-1-dependent inhibition and MEK-ERK activation, the structural basis for non-sialic acid ligand recognition (HMW-HA, prohibitins, viral RBD), and whether therapeutic Siglec-9 blockade or agonism can be translated to clinical benefit in cancer and inflammatory disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No co-crystal structure with a glycoprotein ligand\", \"Mechanism of ligand-dependent signaling mode switching unresolved\", \"Clinical-grade Siglec-9 blocking or agonist agents not yet in trials\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 3, 16, 20, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 5, 20, 24, 31]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 13, 31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 5, 6, 16, 20, 21, 23, 24, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 15, 16, 19, 20]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [31]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SHP-1\",\n      \"MUC16\",\n      \"GYPA\",\n      \"AOC3\",\n      \"MUC1\",\n      \"DSG2\",\n      \"GP1BA\",\n      \"CD59\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}