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1
Zid, Mouldi, and Guy Drouin. "Gene conversions are frequent but not under positive selection in the Siglec gene families of primates." Genome 57, no. 6 (2014): 317–25. http://dx.doi.org/10.1139/gen-2014-0083.
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Siglecs are cell surface proteins that belong to the immunoglobulin superfamily and which bind sialic acids. They are composed of two groups, the conserved Siglecs and the CD33-related Siglecs. Previous studies have reported the occurrence of gene conversions between human CD33-related Siglecs and suggested that these conversions are adaptive because they increase the diversity of these immunoglobulin-related genes. Here, we analyze the Siglec genes of five primate species and show that gene conversions are not observed between conserved Siglec genes but that they are frequent between primate CD33-related Siglecs. The gene conversions between CD33-related Siglec genes only occur between similar genes and equally frequently in sialic acid binding and nonbinding domains. Furthermore, dN/dS ratio tests show that most of the Ig-like V-type 1 and the Ig-like C2-type 1 domains of Siglec genes evolve either neutrally or under purifying selection and that gene conversions were not responsible for the positively selected regions detected in the Ig-like V-type1 domain of the human SIGLEC7 and SIGLEC9 genes. Our results suggest that the frequent gene conversions between CD33-related Siglec genes are simply a consequence of the high degree of sequence similarity of these genes and that they are not adaptive.
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Siddiqui, Shoib Sarwar, Rachel Matar, Maxime Merheb, et al. "Siglecs in Brain Function and Neurological Disorders." Cells 8, no. 10 (2019): 1125. http://dx.doi.org/10.3390/cells8101125.
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Siglecs (Sialic acid-binding immunoglobulin-type lectins) are a I-type lectin that typically binds sialic acid. Siglecs are predominantly expressed in immune cells and generate activating or inhibitory signals. They are also shown to be expressed on the surface of cells in the nervous system and have been shown to play central roles in neuroinflammation. There has been a plethora of reviews outlining the studies pertaining to Siglecs in immune cells. However, this review aims to compile the articles on the role of Siglecs in brain function and neurological disorders. In humans, the most abundant Siglecs are CD33 (Siglec-3), Siglec-4 (myelin-associated glycoprotein/MAG), and Siglec-11, Whereas in mice the most abundant are Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-E, Siglec-F, and Siglec-H. This review is divided into three parts. Firstly, we discuss the general biological aspects of Siglecs that are expressed in nervous tissue. Secondly, we discuss about the role of Siglecs in brain function and molecular mechanism for their function. Finally, we collate the available information on Siglecs and neurological disorders. It is intriguing to study this family of proteins in neurological disorders because they carry immunoinhibitory and immunoactivating motifs that can be vital in neuroinflammation.
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Avril, T., H. Attrill, J. Zhang, A. Raper, and P. R. Crocker. "Negative regulation of leucocyte functions by CD33-related siglecs." Biochemical Society Transactions 34, no. 6 (2006): 1024–27. http://dx.doi.org/10.1042/bst0341024.
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The siglecs (sialic acid-binding Ig-like lectins) are a family of transmembrane receptors expressed in the haemopoietic, immune and nervous systems. The CD33-related siglecs are a distinct subset mostly expressed in the innate immune system where they can function as inhibitory receptors by suppressing the signalling mediated by receptors coupled with ITAMs (immunoreceptor tyrosine-based activation motifs). CD33-related siglecs contain ITIMs (immunoreceptor tyrosine-based inhibitory motifs) that recruit and activate SHP-1 [SH2 (Src homology 2) domain-containing phosphatase-1] and SHP-2. In addition, the ITIMs of CD33-related siglecs can suppress siglec-dependent adhesion of sialylated ligands and mediate endocytosis. Siglec-H is a recently characterized murine CD33-related endocytic receptor that lacks intrinsic tyrosine-based signalling motifs and is expressed selectively on PDCs (plasmacytoid dendritic cells). Siglec-H depends on DAP12 (DNAX-activating protein of 12 kDa) for surface expression and cross-linking with anti-siglec-H antibodies can selectively inhibit interferon-α production by PDCs following TLR9 (Toll-like receptor 9) ligation. Thus CD33-related siglecs are able to mediate diverse inhibitory functions of leucocytes in the innate immune system via both ITIM-dependent and -independent pathways.
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Crocker, Paul R., and Pierre Redelinghuys. "Siglecs as positive and negative regulators of the immune system." Biochemical Society Transactions 36, no. 6 (2008): 1467–71. http://dx.doi.org/10.1042/bst0361467.
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Siglecs (sialic acid-binding Ig-like lectins) are mainly expressed in the immune system. Sn (sialoadhesin) (siglec-1), CD22 (siglec-2) and siglec-15 are well conserved, whereas the CD33-related siglecs are undergoing rapid evolution, as reflected in large differences in repertoires among the different mammals studied so far. In the present paper, we review recent findings on the signalling properties of the CD33-related siglecs and discuss the emergence of both inhibitory and activating forms of this family. We also discuss how Sn may function as a positive regulator of adaptive immune responses and its emerging role as an induced macrophage pattern-recognition molecule for sialylated pathogens, especially enveloped viruses.
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Patra, Madhumita Dandopath. "Structural Studies on Different Ligand Binding Ability of Sialoadhesin Using Molecular Modeling Techniques." Asian Journal of Organic & Medicinal Chemistry 5, no. 4 (2020): 277–82. http://dx.doi.org/10.14233/ajomc.2020.ajomc-p279.
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Siglecs are the major homologous subfamily of I-type lectins with an ability to recognize sialylated glycans. Siglecs are attractive therapeutic targets because of their endocytic properties, ability to modulate receptor signaling and cell-type specific expression pattern. Sialoadhesin (Sn/ Siglec-1/ CD169), a member of the Siglec family expressed on subsets of resident and inflammatory macrophages and involves in modulation of inflammation and immunity. In this work, 3-D structure of human Siglec-1 (hSiglec-1) was predicted based on X-ray crystallo-graphically determined structure of mouse Siglec-1[mSiglec-1(PDB ID: 1QFP)] using molecular modeling techniques. The structure of complexes in solution of hSiglec-1 with ligands, glycopeptide and 3′-sialyllactose were predicted using a novel docking technique comprising of repeated cycles of molecular dynamics and energy minimization. Calculation of the free energies of binding of complexes suggested that glycopeptide can form stable complex with dissociation constant value of 3.31 μM whereas complex formation of 3′-sialyllactose with the protein in aqueous medium is thermodynamically unfavorable. The structural analysis of theses complexes represent the functional recognition interactions of this protein with the bound sugar molecule and as such provide detailed information about functional roles of such sugar binding protein.
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Hayakawa, Toshiyuki, Takashi Angata, Elliott H. Margulies, Tarjei Mikkelsen, Eric D. Green, and Ajit Varki. "Gene Conversion of Sialic Acid Binding Domains in CD33-Related Siglecs by Adjacent Pseudogenes: A Novel Mechanism To Change Sialic Acid Binding Specificity." Blood 104, no. 11 (2004): 1471. http://dx.doi.org/10.1182/blood.v104.11.1471.1471.
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Abstract Siglecs (sialic acid-binding immunoglobulin superfamily lectins) are a family of cell surface receptors involved in regulating the immune response. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules found primarily on cells of the innate immune system. All are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that includes two tyrosine-based signaling motifs. Available data suggest an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. Nine of the 13 known primate Siglec genes along with 14 Siglec pseudogenes comprise the CD33-related Siglec gene cluster on human chromosome 19. Gene conversion is a mechanism for copying part of a genomic sequence into another, contributing to genetic diversity. Pseudogenes are known to play role in generating functional diversity of related genes (e.g., antibody diversity via gene conversion in chickens). We recently analyzed genomic sequences of the CD33-related Siglec gene cluster in three primates (human, chimpanzee and baboon) and found evidence for rapid evolution in this gene family (Angata et al., PNAS, in press). Additional evolutionary studies using distance-based phylogenetic trees shows evidence for three partial gene conversions between Siglec genes and adjacent Siglec pseudogenes. All three involve the coding regions for extracellular domains that mediate sialic acid recognition, and two involve a pseudogene converting a known Siglec gene. Functional analyses using recombinant proteins show marked differences in sialic acid-binding properties between the converted Siglec and its non-converted ortholog. These findings suggest that gene conversion with pseudogenes has contributed to the rapid functional evolution of the Siglecs, and provides a novel mechanism for changing sialic acid binding specificity. We hypothesize that this mechanism allows for rapid evolutionary adjustments in the recognition of endogenous sialic acids as “self”, a potential factor in controlling the innate immune response.
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Trebo, Anna, Nina Ditsch, Tom Degenhardt, et al. "First Evidence for a Role of Siglec-8 in Breast Cancer." International Journal of Molecular Sciences 22, no. 4 (2021): 2000. http://dx.doi.org/10.3390/ijms22042000.
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Sialic acid-binding immunoglobulin-like lectins (Siglecs) are involved in various immune cell-mediated diseases. Their role in cancer is poorly investigated, and research focusses on Siglec-expression on immune cells interacting with tumor cells. This study evaluates the role of Siglec-8 in breast cancer (BC). Siglec-8 expression was analyzed immunohistochemically on 235 primary BC cases and was correlated with clinical and pathological parameters and outcome. Cell culture experiments were performed with various BC cell lines. Siglec-8 was expressed in 215 BC cases and expression was lowest in triple-negative BC. It correlated with estrogen receptor-status, grading and the prognostic factors galectin (Gal)-7 and tumor-associated mucin-1 (TA-MUC1). However, Gal-7 and TA-MUC1 were only prognosticators for clinical outcome in the cohort expressing high (Immunoreactivity score IRS > 3) Siglec-8 levels but not in the low-expressing cohort. Siglec-8 knockdown led to a significantly reduced Gal-7 expression in MCF7 cells. All BC cell lines expressed low Siglec-8-levels, that could be elevated in MCF7 by Peroxisome proliferator-activated receptor (PPARγ)-stimulation. This study demonstrates that Siglec-8 is expressed in BC cells and correlates with known clinical and prognostic parameters. It is probably associated with Gal-7 and TA-MUC1 and might be regulated via PPARγ. Further analyses focusing on functional associations will clarify Siglec-8’s eligibility as a possible therapeutic target.
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YU, Zhenbao, Meryem MAOUI, Liangtang WU, Denis BANVILLE, and Shi-Hsiang SHEN. "mSiglec-E, a novel mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2." Biochemical Journal 353, no. 3 (2001): 483–92. http://dx.doi.org/10.1042/bj3530483.
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The sialic acid-binding immunoglobulin-like lectins (siglecs) represent a recently defined distinct subset of the immunoglobulin superfamily. By using the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase SHP-1 as bait in a yeast two-hybrid screen, we have identified a new member of the mouse siglec family, mSiglec-E. The mSiglec-E cDNA encodes a protein of 467 amino acids that contains three extracellular immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). mSiglec-E is highly expressed in mouse spleen, a tissue rich in leucocytes. The ITIMs of mSiglec-E can recruit SHP-1 and SHP-2, two inhibitory regulators of immunoreceptor signal transduction. This suggests that the function of mSiglec-E is probably an involvement in haematopoietic cells and the immune system as an inhibitory receptor. When expressed in COS-7 cells, mSiglec-E was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that mSiglec-E may function through cell–cell interactions. In comparison with the known members of the siglec family, mSiglec-E exhibits a high degree of sequence similarity to both human siglec-7 and siglec-9. The gene encoding mSiglec-E is localized in the same chromosome as that encoding mouse CD33. Phylogenetic analysis reveals that neither mouse mSiglec-E nor CD33 shows a clear relationship with any human siglecs so far identified.
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Youngblood, Bradford A., John Leung, Rustom Falahati, et al. "Discovery, Function, and Therapeutic Targeting of Siglec-8." Cells 10, no. 1 (2020): 19. http://dx.doi.org/10.3390/cells10010019.
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Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass cell surface receptors that have inhibitory activities on immune cells. Among these, Siglec-8 is a CD33-related family member selectively expressed on human mast cells and eosinophils, and at low levels on basophils. These cells can participate in inflammatory responses by releasing mediators that attract or activate other cells, contributing to the pathogenesis of allergic and non-allergic diseases. Since its discovery in 2000, initial in vitro studies have found that the engagement of Siglec-8 with a monoclonal antibody or with selective polyvalent sialoglycan ligands induced the cell death of eosinophils and inhibited mast cell degranulation. Anti-Siglec-8 antibody administration in vivo to humanized and transgenic mice selectively expressing Siglec-8 on mouse eosinophils and mast cells confirmed the in vitro findings, and identified additional anti-inflammatory effects. AK002 (lirentelimab) is a humanized non-fucosylated IgG1 antibody against Siglec-8 in clinical development for mast cell- and eosinophil-mediated diseases. AK002 administration has safely demonstrated the inhibition of mast cell activity and the depletion of eosinophils in several phase 1 and phase 2 trials. This article reviews the discovery and functions of Siglec-8, and strategies for its therapeutic targeting for the treatment of eosinophil- and mast cell-associated diseases.
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Falahati, Rustom, Jessica Bright, Alejandro Dorenbaum, et al. "A Recombinant Antibody to Siglec-8 Shows Selective ADCC Activity Against Mast Cells from Systemic Mastocytosis Patients." Blood 126, no. 23 (2015): 4092. http://dx.doi.org/10.1182/blood.v126.23.4092.4092.
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Abstract Background: Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by the accumulation of neoplastic mast cells (MC) in one or more extracutaneous organs. In all forms of SM, anti-mediator drugs are used to control symptoms of MC degranulation. In advanced forms of SM, organ damage is common and patients (pts) exhibit reduced life expectancy. In these individuals, cytoreductive agents such as cladribine and interferon-alpha have been used off-label, and inhibitors of KIT D816V are under investigation. A significant unmet need exists for these patients. Siglec-8 is an inhibitory receptor of the CD33-related family of sialic acid-binding, Ig-like lectins (Siglecs) that is expressed selectively on the surface of mature MC, eosinophils, and basophils. Engagement of Siglec-8 with monoclonal antibodies has been previously shown to inhibit IgE-mediated MC degranulation and to induce apoptosis of cytokine-activated eosinophils. Thus this receptor is a potential target for antibody therapy of SM with or without associated eosinophilia. Anti-Siglec-8 antibodies do not directly affect MC viability but antibodies with effector function can induce antibody cell-mediated cytotoxicity (ADCC). Here we show that a recombinant anti-Siglec-8 IgG1 monoclonal antibody can elicit ADCC activity against MC derived from SM patients ex vivo. Methods: Bone marrow (BM) aspirates from SM patients were evaluated for Siglec-8 cell-surface expression on CD117+ FcεRI+ MC or CD25+ MC by flow cytometry. For ADCC assays, BM MC enriched using CD117-targeting magnetic beads or a Siglec-8 transfected Ramos cell line were used as target cells. Peripheral blood leukocytes (PBL) or NK cells purified from peripheral blood were used as effector cells at an effector:target ratio of 10:1. Recombinant anti-Siglec-8 antibody or an isotype control antibody was added at various concentrations and the percent viable CD117+ FcεRI+ MC remaining after 48 hours of culture was determined by flow cytometry. Results: Samples from 9 pts with SM were included in the analysis (ISM, n=1; SSM, n=1; SM-CMML, n=3; SM-MDS, n=1; SM-CEL, n=1; ASM, n=1; MCL, n=1). Eight pts were KIT D816V positive. At the time of sample collection, treatments included midostaurin (n=3); cladribine (n=1); corticosteroids (n=1); and 4 pts were not receiving any biologic or cytoreductive therapy. All BM samples showed detectable CD117+ MC. Robust and selective cell-surface expression of Siglec-8 was observed in all 6 cases evaluated and 100% of CD117+ FcεRI+ MC were Siglec-8 positive by FACS, including CD25+ MC. Levels of Siglec-8 were comparable to or higher than levels on mature MC isolated from normal skin. In this limited sample size, no difference in Siglec-8 expression was observed between patients receiving different therapies or no therapy. To evaluate the ADCC activity of recombinant anti-Siglec-8 antibody on MC, enriched BM MC were incubated with anti-Siglec-8 antibody or isotype-matched control antibody at 1 μg/ mL in the presence of purified NK effector cells. In two patients evaluated, significant anti-Siglec-8-mediated ADCC activity on MC was observed using non-autologous NK cells (69% reduction, 1 pt) or autologous NK cells (76% reduction, 1 pt) indicating that anti-Siglec-8 has the potential to reduce MC burden in these patients. ADCC activity has been reported to be defective in some cancer patients. To evaluate the ability of effector cells in SM patients to mediate ADCC, an assay was developed using a Siglec-8 transfected target cell line to screen blood samples for ADCC activity induced by anti-Siglec-8 antibody. Using PBL as effector cells, ADCC was observed in all samples tested (5/5). Titration of antibody was performed on 2 samples and potent ADCC activity was observed in both, with an EC50 for target depletion of 49 and 65 ng/mL of anti-Siglec-8 antibody, respectively. Conclusion: These data provide a strong rationale for evaluating the effect of an antibody to Siglec-8 with ADCC activity in patients with SM. Disclosures Falahati: Allakos, Inc.: Employment, Other: Options for Equity Owernship. Bright:Allakos, Inc.: Employment, Other: Options for Equity Owernship. Dorenbaum:Allakos, Inc.: Employment, Equity Ownership. Bebbington:Allakos, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tomasevic:Allakos, Inc.: Employment, Equity Ownership. George:Allakos: Research Funding; Novartis: Consultancy. Gotlib:Allakos, Inc.: Consultancy.
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Daly, John, Subhashis Sarkar, Alessandro Natoni, et al. "Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor." Blood 134, Supplement_1 (2019): 138. http://dx.doi.org/10.1182/blood-2019-126613.
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Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (>85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p<0.01 at 0.5:1, p<0.001 at 1:1, p<0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p<0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p<0.05 at 0.5:1, p<0.01 at 1:1) or 3Fax-Neu5Ac (p<0.01 at 0.5:1, p<0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.
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Ruffin, Nicolas, Ester Gea-Mallorquí, Flavien Brouiller, et al. "Constitutive Siglec-1 expression confers susceptibility to HIV-1 infection of human dendritic cell precursors." Proceedings of the National Academy of Sciences 116, no. 43 (2019): 21685–93. http://dx.doi.org/10.1073/pnas.1911007116.
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The human dendritic cell (DC) lineage has recently been unraveled by high-dimensional mapping, revealing the existence of a discrete new population of blood circulating DC precursors (pre-DCs). Whether this new DC population possesses specific functional features as compared to the other blood DC subset upon pathogen encounter remained to be evaluated. A unique feature of pre-DCs among blood DCs is their constitutive expression of the viral adhesion receptor Siglec-1. Here, we show that pre-DCs, but not other blood DC subsets, are susceptible to infection by HIV-1 in a Siglec-1–dependent manner. Siglec-1 mediates pre-DC infection of CCR5- and CXCR4-tropic strains. Infection of pre-DCs is further enhanced in the presence of HIV-2/SIVmac Vpx, indicating that Siglec-1 does not counteract restriction factors such as SAMHD1. Instead, Siglec-1 promotes attachment and fusion of viral particles. HIV-1–infected pre-DCs produce new infectious viral particles that accumulate in intracellular compartments reminiscent of the virus-containing compartment of macrophages. Pre-DC activation by toll-like receptor (TLR) ligands induces an antiviral state that inhibits HIV-1 fusion and infection, but Siglec-1 remains functional and mediates replication-independent transfer of HIV-1 to activated primary T lymphocytes. Altogether, Siglec-1–mediated susceptibility to HIV-1 infection of pre-DCs constitutes a unique functional feature that might represent a preferential relationship of this emerging cell type with viruses.
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Muniz-Trabudua, Xabier, Cristina Borio, Marcos Bilen, et al. "Siglec-1 Expressed on Dendritic Cells is a New Receptor Implicated in Arenavirus Uptake." Proceedings 50, no. 1 (2020): 90. http://dx.doi.org/10.3390/proceedings2020050090.
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Arenaviruses are enveloped viruses that cause hemorrhagic fever outbreaks in humans and still lack an effective antiviral treatment. Upon early infection, these viruses target dendritic cells (DCs), which can promote systemic viral dissemination, contributing to pathogenesis. We have previously described that Siglec-1, a sialic acid Ig-like binding lectin-1 expressed on DCs interacts with different enveloped viruses and promotes their capture within a virus-containing compartment. Such is the case of HIV-1 or Ebola virus, which display sialylated gangliosides on their viral envelope that are effectively recognized by Siglec-1. Here, we aimed to study if Siglec-1 on DCs also interacts with arenaviruses such as Junin. We produced non-infectious Junin viral-like particles (Junin-VLPs) tagged with fluorescent Egfp by transfecting a plasmid encoding the structural Junin Z protein on HEK-293T cells. Junin-VLPs were added to a Raji cell line stably transfected with Siglec-1 or to monocyte-derived DCs activated or not with either Interferon-α or lipopolysaccharide. Viral uptake was analyzed by FACS or confocal microscopy in the presence of an anti-Siglec-1 monoclonal antibody (mAb) or an isotype control. Statistical differences were assessed with the indicated tests. Raji Siglec-1 cells captured a higher number of Junin-VLPs than Raji cells, and this was blocked with an anti-Siglec-1 mAb (P = 0.0159; Mann–Whitney). On primary DCs, activation enhanced Junin-VLP capture (P = 0.0024; paired t-test) and Siglec-1 expression. Furthermore, pre-incubation with an anti-Siglec-1 mAb on activated DCs blocked Junin-VLP uptake (P ≤ 0.0002; one sample t-test), while an isotype control did not. Forty-nine percent of the activated DCs analyzed by confocal microscopy captured Junin-VLPs within a Siglec-1+ virus-containing compartment. Moreover, when HIV-1 was also added, 97% of those compartments retained both viruses. Thus, we conclude that Siglec-1 is a new receptor involved in arenavirus uptake in DCs and could represent a novel target for an anti-arenavirus treatment.
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Virtanen, Helena, Johanna M. U. Silvola, Anu Autio, et al. "Comparison of 68Ga-DOTA-Siglec-9 and 18F-Fluorodeoxyribose-Siglec-9: Inflammation Imaging and Radiation Dosimetry." Contrast Media & Molecular Imaging 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/7645070.
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Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a ligand of inflammation-inducible vascular adhesion protein-1 (VAP-1). We compared 68Ga-DOTA- and 18F-fluorodeoxyribose- (FDR-) labeled Siglec-9 motif peptides for PET imaging of inflammation. Methods. Firstly, we examined 68Ga-DOTA-Siglec-9 and 18F-FDR-Siglec-9 in rats with skin/muscle inflammation. We then studied 18F-FDR-Siglec-9 for the detection of inflamed atherosclerotic plaques in mice and compared it with previous 68Ga-DOTA-Siglec-9 results. Lastly, we estimated human radiation dosimetry from the rat data. Results. In rats, 68Ga-DOTA-Siglec-9 (SUV, 0.88±0.087) and 18F-FDR-Siglec-9 (SUV, 0.77±0.22) showed comparable (P=0.29) imaging of inflammation. In atherosclerotic mice, 18F-FDR-Siglec-9 detected inflamed plaques with a target-to-background ratio (1.6±0.078) similar to previously tested 68Ga-DOTA-Siglec-9 (P=0.35). Human effective dose estimates for 68Ga-DOTA-Siglec-9 and 18F-FDR-Siglec-9 were 0.024 and 0.022 mSv/MBq, respectively. Conclusion. Both tracers are suitable for PET imaging of inflammation. The easier production and lower cost of 68Ga-DOTA-Siglec-9 present advantages over 18F-FDR-Siglec-9, indicating it as a primary choice for clinical studies.
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Wu, Gang, Gavuthami Murugesan, Manjula Nagala, et al. "Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses." Wellcome Open Research 6 (June 1, 2021): 134. http://dx.doi.org/10.12688/wellcomeopenres.16834.1.
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Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple ‘glycogenes’ that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.
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Toubai, Tomomi, Rebecca Evers, Yaping Sun, et al. "CD24-Siglec-G Interaction Plays an Important in Reducing Experimental Graft-Versus-Host Disease (GVHD)." Blood 120, no. 21 (2012): 453. http://dx.doi.org/10.1182/blood.v120.21.453.453.
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Abstract Abstract 453 The role of host antigen presenting cells (APCs) on negatively regulating GVHD is not well understood. Members of the sialic acid binding Ig–like lectin-G (Siglec-G) is an immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in its intracellular domain that negatively regulates immune activation induced by non-infectious damage associated molecules (DAMPs). Following conditioning for allogeneic BMT, several DAMPs are released which stimulate host APCs and enhance GVHD. But the role of negative regulators of DAMP associated immune activation, such as Siglecs, in regulating allo-reactivity is not known. We therefore utilized well defined clinically relevant murine models of allogeneic BMT to test the hypothesis that deficiency of a negative regulator of responses to DAMPs in the hosts, namely Siglec-G, will increase GVHD. B6 wild type (WT) and Siglec-G−/− animals were lethally irradiated (13Gy) and transplanted on day 0 with 5×106 bone marrow and 3×106 splenic CD90+T cells from either syngeneic WT-B6 or MHC mismatched BALB/c donors. The Siglec-G−/− animals showed significantly worse survival than the allo-WT animals (p=0.0045). The increased mortality was associated with an increase in GVHD specific clinical severity (p<0.05), donor T cell expansion (p<0.03), and serum levels of pro-inflammatory cytokines (TNFα, IFN-γ, p<0.05) on day +7 after BMT. We next evaluated whether this was because of Siglec-G deficiency only on the radiosensitive host APCs. To this end we generated [B6àB6Ly5.2] and [Siglec-G−/−àB6Ly5.2] BM chimeras and utilized them as recipients following lethal radiation. They were injected with 5×106 BM and 3×106 CD90+ T cells from either syngeneic WT B6 or allogeneic BALB/c donors. The allogeneic [Siglec-G−/−àB6Ly5.2] animals demonstrated significantly worse survival than the [B6àB6Ly5.2] animals (p<0.0001). We determined the converse, i.e. analyzed the role of Siglec-G on radio-resistant host APCs by generating [B6Ly5.2àB6] and [B6Ly5.2àSiglec-G−/−] BM chimeras and utilized them as recipients. [B6Ly5.2àSiglec-G−/−] chimeras demonstrated similar survival as [B6Ly5.2àB6] chimeras. These data collectively demonstrate that Siglec-G expression only on the host radiosensitive APCs is critical for protection from GVHD. To confirm the role of increased DAMPs in causing greater mortality we tested whether the intensity of conditioning affects the serum level of DAMPs (HMGB1, proinflammatory cytokines) and found that significantly greater levels of DAMPs were observed in the mice that received 13Gy than 8 Gy. Furthermore, consistent with the increased levels of DAMPs, Siglec-G−/− animals showed higher GVHD only after 13Gy radiation but not after 8Gy conditioning. Because responses to non-infectious DAMPs are regulated by Siglec-G through its interaction with CD24 we next hypothesized that enhanced CD24-Siglec-G interaction would mitigate GVHD following myeloablative conditioning. We first characterized stimulation of allogeneic T cell responses by Siglec-G−/− APCs. We utilized CD24−/− and WT BALB/c T cells as responders in an MLR with B6 and Siglec-G−/−stimulators. We found that Siglec-G−/− APCs expanded the CD24−/− T cells more than WT-B6 APCs. We next tested in vivo whether enhanced CD24-Siglec-G interaction would mitigate GVHD. We utilized a novel CD24 fusion protein (day-1, 100mg/mouse) and found that it decreased GVHD mortality only in the WT but not in the Siglec-G−/− animals. Together our data demonstrate a critical role for CD24-Siglec-G interactions in regulating GVHD and suggest that administration of the novel CD24 fusion protein may be an innovative strategy to mitigate GVHD. Disclosures: No relevant conflicts of interest to declare.
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Toubai, Tomomi, Corinne Rossi, Katherine Oravecz-Wilson, et al. "Donor T Cells Intrinsic Responses to Damps Regulated By Siglec-G-CD24 Axis Mitigate Gvhd but Maintain GVL in Experimental BMT Model." Blood 126, no. 23 (2015): 229. http://dx.doi.org/10.1182/blood.v126.23.229.229.
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Abstract Innate immune receptors like pattern recognition receptors (PRRs) including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) like-receptors (NLR) on immune cells play an important role in initiating inflammatory responses to damage- and pathogen- associated molecular patterns (DAMPs and PAMPs) expressed on invading pathogens or released from damaged cells. Although it is well known that DAMPs directly modulate innate immune functions, it is less clear whether DAMPs directly regulate T cell intrinsic function. Members of the sialic acid binding Ig-like lectin (Siglec) family have immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in their intracellular domain that negatively regulate immune activation induced by DAMPs. Our previous data suggested that the Siglec- G-CD24 interaction in host APCs plays an important role in the negative regulation of graft-versus host (GVH) responses. However, the T cell autonomous role of Siglec-G in the regulation of T cell responses is not known. Because Siglecs are important negative regulators of immune responses, we tested the hypothesis that the deficiency of Siglec-G in donor T cells would enhance GVHD. To test our hypothesis, we first examined detailed phenotypic analysis of various T cell subsets and activation markers in naïve Siglec-G-/- and wild-type (WT) B6 animals and found similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of Siglec-G in donors affects GVHD, WT-BALB/cmice were lethally irradiated (850cGy) and transplanted on day 0 with 5x106 bone marrow and 0.5x106 splenic CD90+ T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Siglec-G-/- animals. The recipients receiving donor T cells from Siglec-G-/- animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p<0.05). This increased mortality was also associated with more severe GVHD damage in target organs and a higher expansion of activated CD69+, IFN-r+, and IL-17A+ donor T cells in the spleen and target organs. Enhanced GVHD mortality and severity was also observed in MHC mismatched haploidentical matched B6 in to F1models (p<0.05). To explore the mechanism, we tested whether Siglec-G deficiency alters the naïve T cell responses in vitro after allogeneic or non-specific TCR stimulation in the absence of exogenous DAMPs. Interestingly Siglec-G-/- T cells showed similar proliferation in vitro, when compared to WT B6 T cells. In addition, Siglec-G-/- Tregs are equally suppressive in suppression assay and Siglec-G-/- T cells showed severe GVHD even Tregs are depleted in allo-BMT. However, Siglec-G-/- T cells showed a higher proliferation after direct TCR stimulation (CD3/CD28) with addition of DAMP (HMGB-1) when compared to WT T cells in vitro, suggesting direct T cell intrinsic effects. Consistent with this result, allogeneic Siglec-G-/- T cells caused similar mortality compared to WT controls in non-irradiated B6 into F1 model due to the absence of DAMPs from conditioning. To test the critical cellular mechanisms, we examined the function of endogenous Siglec-G ligand, CD24. We utilized BALB/c CD24-/- animals as hosts in same BMT model and found that CD24-/- animals showed an enhanced GVHD mortality and severity when compared to WT animals (p<0.05). To enhance Siglec-G-CD24 axis, we utilized a novel CD24 fusion protein (CD24Fc) in same BMT model and found that CD24 Fc ameliorated GVHD severity and mortality in not only allogeneic WT-B6 animals (p<0.05) but also CD24-/- animals (p<0.05). Next we explored DAMPs regulation by Siglec-G-CD24 axis in GVL. We utilized the same model of CD24Fc treatment but added P815 at the same time of allo-BMT and found that CD24Fc treated animals showed equivalent GVL to non-treated animals, suggesting that regulation of DAMPs with CD24Fc mitigates GVHD with maintaining GVL effect. Collectively our data suggested that the expression of both Siglec-G on donor T cells and CD24 on hosts is critical for controlling GVHD in the context of DAMPs released from conditioning, and represents a novel strategy that CD24Fc can mitigates GVHD with maintaining GVL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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Bibollet-Ruche, Frederic, Brett A. McKinney, Alexandra Duverger, Frederic H. Wagner, Aftab A. Ansari, and Olaf Kutsch. "The Quality of Chimpanzee T-Cell Activation and Simian Immunodeficiency Virus/Human Immunodeficiency Virus Susceptibility Achieved via Antibody-Mediated T-Cell Receptor/CD3 Stimulation Is a Function of the Anti-CD3 Antibody Isotype." Journal of Virology 82, no. 20 (2008): 10271–78. http://dx.doi.org/10.1128/jvi.01319-08.
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ABSTRACT While human immunodeficiency virus type 1 (HIV-1) infection is associated with hyperimmune activation and systemic depletion of CD4+ T cells, simian immunodeficiency virus (SIV) infection in sooty mangabeys or chimpanzees does not exhibit these hallmarks. Control of immune activation is thought to be one of the major components that govern species-dependent differences in the disease pathogenesis. A previous study introduced the idea that the resistance of chimpanzees to SIVcpz infection-induced hyperimmune activation could be the result of the expression of select sialic acid-recognizing immunoglobulin (Ig)-like lectin (Siglec) superfamily members by chimpanzee T cells. Siglecs, which are absent on human T cells, were thought to control levels of T-cell activation in chimpanzees and were thus suggested as a cause for the pathogenic differences in the course of SIVcpz or HIV-1 infection. As in human models of T-cell activation, stimulation had been attempted using an anti-CD3 monoclonal antibody (MAb) (UCHT1; isotype IgG1), but despite efficient binding, UCHT1 failed to activate chimpanzee T cells, an activation block that could be partially overcome by MAb-induced Siglec-5 internalization. We herein demonstrate that anti-CD3 MAb-mediated chimpanzee T-cell activation is a function of the anti-CD3 MAb isotype and is not governed by Siglec expression. While IgG1 anti-CD3 MAbs fail to stimulate chimpanzee T cells, IgG2a anti-CD3 MAbs activate chimpanzee T cells in the absence of Siglec manipulations. Our results thus imply that prior to studying possible differences between human and chimpanzee T-cell activation, a relevant model of chimpanzee T cell activation needs to be established.
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Alkhodair, K., H. Almhanna, J. McGetrick, et al. "Siglec expression on the surface of human, bull and ram sperm." Reproduction 155, no. 4 (2018): 361–71. http://dx.doi.org/10.1530/rep-17-0475.
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Sialic acid (Sia) is a major constituent of both the sperm glycocalyx and female reproductive mucosal surface and is involved in regulating sperm migration, uterotubal reservoir formation and oocyte binding. Siglecs (sialic acid-binding immunoglobulin – like lectins) commonly found on immune cells, bind to Sia in a linkage- and sugar-specific manner and often mediate cell-to-cell interactions and signalling. Proteomic and transcriptomic analysis of human and bovine sperm have listed Siglecs, but to date, their presence and/or localisation on sperm has not been studied. Therefore, the aim of this study was to characterise the presence of Siglecs on the surface of bovine, human and ovine sperm using both immunostaining and Western blotting. Siglec 1, 2, 5, 6, 10 and 14 were identified and displayed both species- and regional-specific expression on sperm. Almost universal expression across Siglecs and species was evident in the sperm neck and midpiece region while variable expression among Siglecs, similar among species, was detected in the head and tail regions of the sperm. The possible role for these proteins on sperm is discussed.
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Kivi, Elina, Kati Elima, Kristiina Aalto, et al. "Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate." Blood 114, no. 26 (2009): 5385–92. http://dx.doi.org/10.1182/blood-2009-04-219253.
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Abstract Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10–VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.
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Brunetta, Enrico, Manuela Fogli, Stefania Varchetta, et al. "The decreased expression of Siglec-7 represents an early marker of dysfunctional natural killer–cell subsets associated with high levels of HIV-1 viremia." Blood 114, no. 18 (2009): 3822–30. http://dx.doi.org/10.1182/blood-2009-06-226332.
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Abstract HIV-1 has developed several strategies to evade natural killer (NK)–cell antiviral functions. One of these mechanisms is the HIV-1–induced expansion of highly dysfunctional NK-cell subsets. Here, we analyze a large cohort of HIV-1–infected patients in early or chronic phases of infection, both cross-sectionally and longitudinally. We demonstrate that a striking decrease in the surface expression of sialic acid–binding immunoglobulin-like lectin 7 (Siglec-7) represents the earliest marker of the aberrant NK-cell dysregulation, which precedes the down-modulation of CD56 mostly occurring in patients with chronic HIV-1 viremia. The combined detection of Siglec-7 and CD56 allows the identification of 2 new pathologic NK-cell subsets expanded preferentially in early (Siglec-7−/CD56+) or chronic (Siglec-7−/CD56−) stages of HIV-1 infection. Remarkably, these phenotypic abnormalities were directly associated with progressive and distinct impairments of NK-cell functions. The aforementioned NK-cell aberrancies could be observed only in the presence of high levels of viral replication and not in patients with low or undetectable HIV-1 viremia, such as long-term nonprogressors or patients having undergone antiretroviral therapy. High frequencies of Siglec-7−/CD56+ and Siglec-7−/CD56− pathologic NK cells reflect the immune and clinical status of HIV-1 infection and can also track the effectiveness of therapy.
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Aalto, Kristiina, Anu Autio, Elina A. Kiss, et al. "Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer." Blood 118, no. 13 (2011): 3725–33. http://dx.doi.org/10.1182/blood-2010-09-311076.
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Abstract Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.
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Chrusciel, Paulina, Emrah Yatkin, Xiang-Guo Li, et al. "Safety Study of Single-Dose Intravenously Administered DOTA-Siglec-9 Peptide in Sprague Dawley Rats." International Journal of Toxicology 38, no. 1 (2019): 4–11. http://dx.doi.org/10.1177/1091581818821606.
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The peptide-based radioactive compound [68Ga]Ga-DOTA-Siglec-9 is a novel agent for imaging of inflammation with positron emission tomography. The drug target of [68Ga]Ga-DOTA-Siglec-9 is vascular adhesion protein 1. Previous studies have obtained promising results with [68Ga]Ga-DOTA-Siglec-9 in experimental animals. However, before taking this novel imaging agent into clinical trials, safety and toxicological studies need to be performed with the nonradioactive precursor compound DOTA-Siglec-9. This extended single-dose toxicity study was designed to provide information on the major toxic effects of DOTA-Siglec-9 and to indicate possible target organs after a single intravenous (iv) injection in rats. The study was performed using 60 adult Hsd: Sprague Dawley rats and included a control group and a treatment group to investigate the toxicity of DOTA-Siglec-9 solution at a final concentration of 0.2 mg/mL after a single iv injection of 582 µg/kg. The maximum dose tested was 1,000-fold the clinical dose on a mg/kg basis as indicated in European Medicines Agency International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guideline M3(R2). The planned human clinical dose is approximately 0.582 µg of DOTA-Siglec-9 per kg of body mass. This study demonstrates that iv administration of DOTA-Siglec-9 at a dose of 582 µg/kg was well tolerated in rats and did not produce toxicologically significant adverse effects.
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Jødal, Lars, Anne Roivainen, Vesa Oikonen, et al. "Kinetic Modelling of [68Ga]Ga-DOTA-Siglec-9 in Porcine Osteomyelitis and Soft Tissue Infections." Molecules 24, no. 22 (2019): 4094. http://dx.doi.org/10.3390/molecules24224094.
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Background: [68Ga]Ga-DOTA-Siglec-9 is a positron emission tomography (PET) radioligand for vascular adhesion protein 1 (VAP-1), a protein involved in leukocyte trafficking. The tracer facilitates the imaging of inflammation and infection. Here, we studied the pharmacokinetic modelling of [68Ga]Ga-DOTA-Siglec-9 in osteomyelitis and soft tissue infections in pigs. Methods: Eight pigs with osteomyelitis and soft tissue infections in the right hind limb were dynamically PET scanned for 60 min along with arterial blood sampling. The fraction of radioactivity in the blood accounted for by the parent tracer was evaluated with radio-high-performance liquid chromatography. One- and two-tissue compartment models were used for pharmacokinetic evaluation. Post-mortem soft tissue samples from one pig were analysed with anti-VAP-1 immunofluorescence. In each analysis, the animal’s non-infected left hind limb was used as a control. Results: Tracer uptake was elevated in soft tissue infections but remained low in osteomyelitis. The kinetics of [68Ga]Ga-DOTA-Siglec-9 followed a reversible 2-tissue compartment model. The tracer metabolized quickly; however, taking this into account, produced more ambiguous results. Infected soft tissue samples showed endothelial cell surface expression of the Siglec-9 receptor VAP-1. Conclusion: The kinetics of [68Ga]Ga-DOTA-Siglec-9 uptake in porcine soft tissue infections are best described by the 2-tissue compartment model.
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Bandala-Sanchez, Esther, Naiara G. Bediaga, Ethan D. Goddard-Borger, et al. "CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function." Proceedings of the National Academy of Sciences 115, no. 30 (2018): 7783–88. http://dx.doi.org/10.1073/pnas.1722056115.
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CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.
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Sakamoto, Yuzuru, Sachiyo Yoshio, Akihisa Nagatsu, et al. "Increased frequency of PD-1+CD57+Siglec-7- dysfunctional NK cells in patients with nonalcoholic fatty liver disease." Journal of Clinical Oncology 38, no. 4_suppl (2020): 589. http://dx.doi.org/10.1200/jco.2020.38.4_suppl.589.
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589 Background: The proportion of non-alcoholic fatty liver disease (NAFLD) has been increasing as a cause of hepatocellular carcinoma (HCC) worldwide. Natural killer (NK) cells are involved in the first line of immune defense against cancer. NK cell function is regulated by activating and inhibitory NK cell receptors. However, the role of NK cells in the pathogenesis of NAFLD and NAFLD-HCC is still largely unknown. In this study, we aimed to clarify the phenotypic and functional features of NK cells in NAFLD and NAFLD-HCC patients. Methods: We performed mass cytometry (CyTOF) and flow cytometry analysis of NK cells in 33 NAFLD patients (22 chronic hepatitis (CH), 8 liver cirrhosis (LC), 3 with HCC (HCC)) and 9 healthy donors (HDs). We compared surface markers on NK cells in cancerous and non-cancerous intrahepatic lymphocytes (IHLs). We also measured NK cell function in the presence of IL-12 and IL-18. Results: The frequency of NK cells was lower in NAFLD patients compared to HDs. PD-1, CD57, ILT2 were highly expressed, and Siglec-7, NKp30, NKp46 were downregulated on NK cells from NAFLD patients, compared with those of HDs. In NAFLD patients, Siglec-7 levels on NK cells were negatively correlated with PD-1 and CD57, and positively correlated with NKp30 and NKp46. The other inhibitory markers (NKG2A, KIR3DL1 and KIRDL2/L3), activating markers (CD69 and NKG2D) and checkpoint markers (Tim-3 and TIGIT) were comparable between NAFLD patients and HDs. PD-1 and CD57 expression levels on NK cells were also significantly upregulated in NAFLD-HCC patients than those in HDs. CD57 was rarely expressed on NK cells in non-cancerous IHLs, on the other hand, highly expressed in cancerous IHLs. The IFNγ production and CD107a expression on NK cells were also decreased in NAFLD patients. PD-1+CD57+Siglec-7− NK cells were observed in NAFLD patients, rarely in HDs. PD-1+CD57+Siglec-7− NK cells were functionally impaired compared to other NK subsets. Conclusions: In patients with NAFLD, NK cells are reduced and functionally impaired, the reason of which may be the increased proportion of dysfunctional PD-1+CD57+Siglec-7− NK subset, and dysfunctional NK cells might be related to impairment of surveillance for HCC.
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Perez-Zsolt, Daniel, Javier Martinez-Picado, and Nuria Izquierdo-Useros. "When Dendritic Cells Go Viral: The Role of Siglec-1 in Host Defense and Dissemination of Enveloped Viruses." Viruses 12, no. 1 (2019): 8. http://dx.doi.org/10.3390/v12010008.
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Dendritic cells (DCs) are among the first cells that recognize incoming viruses at the mucosal portals of entry. Initial interaction between DCs and viruses facilitates cell activation and migration to secondary lymphoid tissues, where these antigen presenting cells (APCs) prime specific adaptive immune responses. Some viruses, however, have evolved strategies to subvert the migratory capacity of DCs as a way to disseminate infection systemically. Here we focus on the role of Siglec-1, a sialic acid-binding type I lectin receptor potently upregulated by type I interferons on DCs, that acts as a double edge sword, containing viral replication through the induction of antiviral immunity, but also favoring viral spread within tissues. Such is the case for distant enveloped viruses like human immunodeficiency virus (HIV)-1 or Ebola virus (EBOV), which incorporate sialic acid-containing gangliosides on their viral membrane and are effectively recognized by Siglec-1. Here we review how Siglec-1 is highly induced on the surface of human DCs upon viral infection, the way this impacts different antigen presentation pathways, and how enveloped viruses have evolved to exploit these APC functions as a potent dissemination strategy in different anatomical compartments.
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Linnartz, Bettina, Yiner Wang, and Harald Neumann. "Microglial Immunoreceptor Tyrosine-Based Activation and Inhibition Motif Signaling in Neuroinflammation." International Journal of Alzheimer's Disease 2010 (2010): 1–7. http://dx.doi.org/10.4061/2010/587463.
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Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM-) Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2), signal regulatory protein-1, and complement receptor-3 (CD11b/CD18) signal via the adaptor protein DAP12 and activate phagocytic activity of microglia. Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- (ITIM-) signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec-11 with sialic acids on the neuronal glycocalyx. Thus, ITAM- and ITIM-signaling receptors modulate microglial phagocytosis and cytokine expression during neuroinflammatory processes. Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.
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Zorn-Pauly, L., A. S. L. Von Stuckrad, J. Klotsche, et al. "POS0744 A NEGATIVE INTERFERON BIOMARKER CD169 / SIGLEC-1 RULES OUT SYSTEMIC LUPUS ERYTHEMATOSUS." Annals of the Rheumatic Diseases 80, Suppl 1 (2021): 623.2–624. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2096.
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Background:While there have been advances in the therapy of systemic lupus erythematosus (SLE) in recent years, there have been no major new findings in SLE biomarkers [1, 2]. Type I interferon (IFN) plays a pivotal role in the pathogenesis of SLE [3]. In 2008, we first described CD169 / SIGLEC-1 (sialic acid-binding immunoglobulin-like lectin-1), an interferon-induced adhesion molecule on monocytes in SLE patients [4]. For over five years SIGLEC-1 has been routinely assessed in our clinic.Objectives:To evaluate and compare the diagnostic utility of the type I IFN induced SIGLEC-1 with established biomarkers in the initial diagnosis of the disease.Methods:We analyzed retrospectively 232 patients who were on suspicion of SLE at Charité University Hospital Berlin between October 2015 and September 2020. Patients underwent full clinical characterization, and biomarkers were determined in the routine laboratory. Based on the final diagnosis, we divided patients into two groups: A) initial diagnosis of SLE and B) Non-SLE mimicking condition.Results:In 76 patients (32.3 %) SLE was confirmed by fulfilling the EULAR / ACR 2019 classification criteria [5]. SIGLEC-1 was dramatically increased in patients with an initial diagnosis of SLE compared to patients without SLE (p<0.0001). For a threshold of 2500 molecule per monocyte, a sensitivity of 98.7 %, a specificity of 82.1 %, a negative predictive value (NPV) of 99.2 %, and a positive predictive value (PPV) of 72.8 % were calculated for SIGLEC-1. Adjusted to the prevalence of SLE in Germany (36.7 per 100,000 inhabitants [6]) NPV and PPV turned out to > 99.9 % and 0.2 %. We further aimed to compare not only the performance of the tests at a given cutoff but also across all possible measured values. Therefore, we conducted ROC curves analyses (see figure 1). The area under the curve (AUC) of SIGLEC-1 test was significantly higher than that of ANA test (AUC=0.88, p=0.031), C3 (AUC = 0.83, p=0.001), C4 (AUC=0.83, p=0.002), but not than that of the Anti-dsDNA ELISA (AUC=0.90, p=0.163).Conclusion:Our study shows that IFN activity is a hallmark at the onset of the disease and that the interferon biomarker SIGLEC-1 is valuable to rule out SLE in suspected cases.References:[1]Ostendorf L, Burns M, Durek P, Heinz GA, Heinrich F, Garantziotis P, Enghard P, Richter U, Biesen R, Schneider U et al: Targeting CD38 with Daratumumab in Refractory Systemic Lupus Erythematosus. N Engl J Med 2020, 383(12):1149-1155.[2]Furie R, Rovin BH, Houssiau F, Malvar A, Teng YKO, Contreras G, Amoura Z, Yu X, Mok CC, Santiago MB et al: Two-Year, Randomized, Controlled Trial of Belimumab in Lupus Nephritis. N Engl J Med 2020, 383(12):1117-1128.[3]Ronnblom L, Leonard D: Interferon pathway in SLE: one key to unlocking the mystery of the disease. Lupus Sci Med 2019, 6(1):e000270.[4]Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus M, Haupl T, Rudwaleit M, Riemekasten G, Radbruch A et al: Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum 2008, 58(4):1136-1145.[5]Aringer M, Costenbader K, Daikh D, Brinks R, Mosca M, Ramsey-Goldman R, Smolen JS, Wofsy D, Boumpas DT, Kamen DL et al: 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Annals of the Rheumatic Diseases 2019, 78(9):1151-1159.[6]Brinks R, Fischer-Betz R, Sander O, Richter JG, Chehab G, Schneider M: Age-specific prevalence of diagnosed systemic lupus erythematosus in Germany 2002 and projection to 2030. Lupus 2014, 23(13):1407-1411.Disclosure of Interests:None declared
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Resa-Infante, Patricia, Itziar Erkizia, Jon Ander Nieto-Garai, Maier Lorizate, Nuria Izquierdo-Useros, and Javier Martinez-Picado. "Novel Methodology for the Detection of Enveloped Viruses." Proceedings 50, no. 1 (2020): 52. http://dx.doi.org/10.3390/proceedings2020050052.
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Viral infections in humans cause a huge burden in worldwide healthcare that has increased due to the emergence of new pathogenic viruses, such as in the recent Ebola virus (EBOV) outbreaks. Viral particles in body fluids are often at very low levels, making diagnosis difficult. In order to address this problem, we have developed a new detection platform to isolate and detect different enveloped viruses. We have recently identified that sialic acid-binding Ig‑like lectin 1 (Siglec-1/CD169) is one cellular receptor used by EBOV and HIV-1 to enter myeloid cells, key target cells for infection and pathogenesis. For viral uptake, the V-set domain of this myeloid cell receptor recognizes the gangliosides of viral membranes that were dragged during viral budding from the plasma membrane of infected cells. We took advantage of this specific interaction between Siglec‑1 and viral gangliosides to develop a new detection methodology. We have generated a recombinant protein that contains the V-set domain of Siglec-1 fused to the human IgG Fc domain for anchoring in latex beads. These coated beads allow the isolation of viral particles and their measurement by flow cytometry. We have tested its efficacy to detect HIV-1 and EBOV and its specificity by using anti-Siglec‑1 antibodies that prevent the interaction and serve as a negative control. To test the capacity of our method, we used synthetic liposomes to assess the effect of ganglioside concentration in membranes as well as the size of viral particles. This methodology would facilitate the diagnosis of infections by concentrating viral particles in a fast and direct method. At a time when global human mobility facilitates the dissemination of infectious agents, our approach represents a rapid and effective method to maximize the identification of both known and emerging enveloped viruses as part of public health viral surveillance strategies.
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Eakin, Amanda J., Michael J. Bustard, Cathy M. McGeough, Tahanver Ahmed, Anthony J. Bjourson, and David S. Gibson. "Siglec-1 and -2 as potential biomarkers in autoimmune disease." PROTEOMICS - Clinical Applications 10, no. 6 (2016): 635–44. http://dx.doi.org/10.1002/prca.201500069.
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Kitzig, Friederike, Águeda Martinez-Barriocanal, Miguel López-Botet, and Joan Sayós. "Cloning of two new splice variants of Siglec-10 and mapping of the interaction between Siglec-10 and SHP-1." Biochemical and Biophysical Research Communications 296, no. 2 (2002): 355–62. http://dx.doi.org/10.1016/s0006-291x(02)00885-9.
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Egan, Hannah, Oliver Treacy, Kevin Lynch, et al. "865 Sugar high: Does the sialic acid profile of cancer-associated fibroblasts induce a more tumour-permissive microenvironment?" Journal for ImmunoTherapy of Cancer 8, Suppl 3 (2020): A918. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0865.
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BackgroundImmunosuppressive tumour microenvironments (TME) inhibit the effectiveness of cancer immunotherapies. Sialic acids, which exist as terminal sugars of glyco-conjugates, are highly expressed on cancer cells and are involved in various pathological processes including increased immune evasion, tumour invasiveness and tumour cell metastasis.1 Siglecs (Sialic acid-binding immunoglobulin-type lectins) are expressed on immune cell surfaces and bind sialic acid. Siglec binding to hypersialylated tumour glycans blocks immune cell activation to promote immunosuppression.1 2Intestinal stromal cells (iSCs), precursors to cancer-associated fibroblasts (CAFs), are a key component of the TME and play a vital role in tumour progression by enhancing a tumour-promoting microenvironment. The aim of this study was therefore to investigate if iSC/CAF sialylation contributes to enhanced immunosuppression in the TME.Methods iSCs were isolated from colorectal cancer patient biopsies and cultured ex vivo. Informed consent was obtained from all patients prior to sampling. Tumour-derived iSCs were termed CAFs while control iSCs, isolated from tumour-adjacent non-cancerous tissue, were termed normal-associated fibroblasts (NAFs). NAFs/CAFs were then co-cultured with healthy allogeneic PBMCs and their immunosuppressive properties were assessed by flow cytometry.ResultsCAFs significantly supressed the proliferation of CD8+ and CD4+ T-cells and induced a more exhausted T-cell phenotype as evidenced by increased expression of the exhaustion markers TIM-3, LAG-3 and PD-1 when compared to co-culture with control NAFs, thereby demonstrating their potent immunosuppressive properties. Strikingly, CAFs also induced significantly higher expression of both Siglec-7 and Siglec-9 receptors on CD8+ T-cells specifically.To elucidate the role of sialylation on CAF-mediated immunosuppression, NAFs/CAFs were treated with the sialyltransferase inhibitor (SI) P-3FAX-Neu5Ac prior to co-culture. Reduction of sialic acid expression on NAFs/CAFs was confirmed by flow cytometry and the SI-treated NAFs/CAFs were then co-cultured with allogeneic T-cells to assess the functional consequences of reduced NAF/CAF sialylation. SI-treated CAFs induced significantly less CD4+TIM-3+ and both CD4+LAG-3+ and CD8+LAG-3+ T-cells compared to their untreated counterparts. Interestingly, SI-treated CAFs also induced significantly less Siglec-7 and -9 receptor-expressing CD8+ T-cells.ConclusionsThese results demonstrate that non-haematopoietic stromal cells in the tumour-microenvironment can suppress activated T-cells and that this immunosuppressive effect can be significantly reversed through the modulation of sialylation on the stromal cell surface. These results support the hypothesis that stromal cell sialylation plays a role in their immunosuppressive properties. Understanding how sialylation of stromal cells is regulated and functions to enhance immunosuppression in the TME could uncover novel immune checkpoints to reactivate anti-tumour immunity, allowing for tumour cell clearance.Ethics ApprovalThis study was approved by Galway University Hospitals’ Clinical Research Ethics Committee, approval number C.A 2074.ConsentN/AReferencesWang L, Liu Y, Wu L, Sun XL. Sialyltransferase inhibition and recent advances. Biochim Biophys Acta 2016 Jan; 1864(1):143-53.Munkley J, Scott E. Targeting aberrant sialylation to treat cancer. Medicines (Basel) 2019 Oct 13;6(4):102.
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Li, Xiang-Guo, Anu Autio, Helena Ahtinen, et al. "Translating the concept of peptidelabeling with 5-deoxy-5-[18F]fluororibose into preclinical practice: 18F-labeling of Siglec-9 peptide for PET imaging of inflammation." Chemical Communications 49, no. 35 (2013): 3682–84. http://dx.doi.org/10.1039/c3cc40738a.
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Kai, Xin, Vasant Chellappa, Jesse Moya, et al. "Defective Inhibitory Signaling in CLL B Cells and Increased Recruitment of PI3K by c-Cbl in Zap-70+ CLL." Blood 114, no. 22 (2009): 1258. http://dx.doi.org/10.1182/blood.v114.22.1258.1258.
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Abstract Abstract 1258 Poster Board I-280 Background An inhibitory signaling pathway involving sialic acid 9-O-acetyl esterase (SIAE), sialic acid binding lectins (Siglecs) particularly Siglec-2/CD22, the Lyn tyrosine kinase, and the SH2 domain containing tyrosine phosphatase, SHP-1, attenuates B cell receptor signaling and sets a threshold for B cell activation. A key step in the process is the requirement that SIAE access N-glycans on Siglec ligands and remove 9-O-acetyl groups from terminal αa2,6 linked sialic acid moieties. Siglec-ligand interaction is followed by phosphorylation of ITIM tyrosines on CD22 by Lyn, and the recruitment of SHP-1 by CD22 resulting in signal attenuation (Cariappa et al., J.Exp.Med.2009, 206, 125). While Lyn has both positive and negative signaling functions, knockout mice studies suggest that inhibitory functions are dominant. Previous studies have shown that CLL cells overexpress active Lyn at the protein level and that Lyn is localized to sites beyond the plasma membrane. Although cell surface expression of CD22 is reduced in CLL, it is not known if CD22 can be accessed in CLL cells by promiscuously active Lyn. We sought to ask if cancer progression in CLL involves the evolution of mechanisms to evade inhibitory signaling, thus tipping the balance towards positive, pro-proliferative signaling by Lyn. Methods CLL B cells from patient and control subjects were isolated. Immunoprecipitation and Western blot approaches were used to quantitate the total cellular levels of Lyn and CD22αa and β proteins at the protein level, the ratio of CD22 phosphorylated on an inhibitory tyrosine to total CD22, recruitment of SHP-1 by CD22, the activation of Syk, the expression of c-Cbl and the recruitment of PI3K by c-Cbl in CLL and control B cells. Results A modest decrease in total CD22αa and β proteins was observed in CLL but a dramatic reduction in the proportion of ITIM-phosphorylated CD22, and a reduction in the recruitment of SHP-1 by CD22 in CLL B cells. Decreased inhibitory signaling in CLL correlates with an increase in active Syk. An increase in c-Cbl protein levels was observed and an increased recruitment of p85PI3K was observed specifically in Zap-70 positive CLL. Conclusions Defective inhibitory signaling may contribute to disease progression in CLL. This defect probably results from the inability of CD22 to access 9-O-deacetylated ligands even in the presence of active Lyn. Enhanced constitutive BCR signaling prevails in all CLL patients but in Zap70+ CLL patients p85PI3K is more readily recruited by c-Cbl. Disclosures Hochberg: Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau; Enzon: Speakers Bureau.
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Käkelä, Meeri, Pauliina Luoto, Tapio Viljanen, et al. "Adventures in radiosynthesis of clinical grade [68Ga]Ga-DOTA-Siglec-9." RSC Advances 8, no. 15 (2018): 8051–56. http://dx.doi.org/10.1039/c7ra12423f.
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[<sup>68</sup>Ga]Ga-DOTA-Siglec-9 is the first vascular adhesion protein-1 targeting radiopharmaceutical for positron emission tomography imaging of inflammation, and here we present its long-awaited clinical grade radiosynthesis.
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Winn, Virginia D., Matthew Gormley, Agnes C. Paquet, et al. "Severe Preeclampsia-Related Changes in Gene Expression at the Maternal-Fetal Interface Include Sialic Acid-Binding Immunoglobulin-Like Lectin-6 and Pappalysin-2." Endocrinology 150, no. 1 (2008): 452–62. http://dx.doi.org/10.1210/en.2008-0990.
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Preeclampsia (PE), which affects 4–8% of human pregnancies, causes significant maternal and neonatal morbidity and mortality. Within the basal plate, placental cytotrophoblasts (CTBs) of fetal origin invade the uterus and extensively remodel the maternal vasculature. In PE, CTB invasion is often shallow, and vascular remodeling is rudimentary. To better understand possible causes, we conducted a global analysis of gene expression at the maternal-fetal interface in placental samples from women with PE (n = 12; 24–36 wk) vs. samples from women who delivered due to preterm labor with no evidence of infection (n = 11; 24–36 wk), a condition that our previous work showed is associated with normal CTB invasion. Using the HG-U133A&B Affymetrix GeneChip platform, and statistical significance set at log odds-ratio of B &gt;0, 55 genes were differentially expressed in PE. They encoded proteins previously associated with PE [e.g. Flt-1 (vascular endothelial growth factor receptor-1), leptin, CRH, and inhibin] and novel molecules [e.g. sialic acid binding Ig-like lectin 6 (Siglec-6), a potential leptin receptor, and pappalysin-2 (PAPP-A2), a protease that cleaves IGF-binding proteins]. We used quantitative PCR to validate the expression patterns of a subset of the genes. At the protein level, we confirmed PE-related changes in the expression of Siglec-6 and PAPP-A2, which localized to invasive CTBs and syncytiotrophoblasts. Notably, Siglec-6 placental expression is uniquely human, as is spontaneous PE. The functional significance of these novel observations may provide new insights into the pathogenesis of PE, and assaying the circulating levels of these proteins could have clinical utility for predicting and/or diagnosing PE. Gene expression analysis of placental basal plates from severe preeclamptic pregnancies and controls revealed differential expression of 55 genes, including Siglec-6 and pappalysin-2.
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Kiser, Zachary Monroe, Greta L. Becker, Julia Nguyen, et al. "Decreased Erythrocyte Binding Capability for Neutrophil Siglec-9 Is a Source of Oxidative Stress in Sickle Cell Disease." Blood 132, Supplement 1 (2018): 3650. http://dx.doi.org/10.1182/blood-2018-99-113579.
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Abstract Introduction Oxidative stress and inflammation promote hemolysis and vaso-occlusion in sickle cell disease (SCD). Erythrocytes can play a pro-oxidative and anti-oxidative role in disease-associated inflammation through iron-driven free radical production and endogenous anti-oxidants. In SCD, HbS accelerated auto-oxidation, iron de-compartmentalization, and inflammatory cell-derived oxidants drive this oxidative stress. CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids in "Self-Associated Molecular Patterns" (SAMPs) and typically inhibit innate immune cell functions via cytosolic signaling. Recent studies have shown that Siglec-9 on human neutrophils in circulating blood interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactivity, including reactive oxygen species (ROS) production. Modification of erythrocyte membrane sialic acids interferes with their ability to inhibit neutrophil activation and oxidative burst. As erythrocytes age and undergo cellular damage there is a loss of membrane bound sialoglycoproteins. Several studies have indicated that this reduction in the sialome is accelerated on the sickle erythrocyte. We hypothesize that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and that decreased binding of neutrophil Siglec-9 to sickle erythrocyte sialic acid enhances neutrophil activation and oxidative burst. Methods & Results Binding of recombinant Siglec-9-Fc protein to AA (n=9) and SS erythrocytes (n=7) was measured using flow cytometry. SS erythrocytes displayed significantly less Siglec-9-Fc binding 45% ± 11.9 (mean ± SEM) compared to AA erythrocytes 82% ± 5.4 (p=.03). Treatment of AA erythrocytes with neuraminidase to remove sialic acid decreased binding to 4% ± 7.9. Neutrophil ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Neutrophils were purified from AA donors (n=5) and SS (n=11) or AA erythrocytes (n=5) were added to the neutrophils at a ratio of 50 erythrocytes to 1 neutrophil. Oxidative burst was stimulated using phorbol 12-myristate 13-acetate (PMA). AA erythrocytes decreased PMA-stimulated neutrophil ROS production by 84% ± 6.7. In contrast, SS erythrocytes decreased PMA-stimulated neutrophil ROS production by 53% ± 6.8 (p=0.03,). Recent studies have shown that neutrophil extracellular trap (NET) formation is pathogenic in SCD. We added AA and SS erythrocytes (50:1) to neutrophils stimulated with PMA and NET formation was assessed using Syto Orange, Syto Green and confocal microscopy. PMA-stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation. In contrast, AA erythrocytes treated with neuraminidase to remove sialic acid had increased NET formation. PMA-stimulated neutrophils incubated with SS erythrocytes showed increased NET formation. Conclusions A constant disease state of oxidative stress and inflammation underlies SCD pathophysiology. These data demonstrate that SS erythrocytes with decreased membrane sialic acid are deficient in binding to neutrophil Siglec-9. The decreased binding of SS erythrocytes to neutrophil Siglec-9 diminishes the ability of SS erythrocytes to properly modulate neutrophil activation, which may contribute to the oxidative stress and increased state of basal inflammation inherent to SCD. The overall reduction in number of RBCs in SS may also be a factor? Figure. Figure. Disclosures Belcher: CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding.
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Youngblood, Bradford A., Emily C. Brock, John Leung, Alan T. Chang, Christopher Bebbington, and Nenad Tomasevic. "AK002, a Novel Humanized Monoclonal Antibody to Siglec-8, Inhibits Mast Cell Activity and Depletes Eosinophils in Ex Vivo Bone Marrow Tissue from Patients with Systemic Mastocytosis." Blood 132, Supplement 1 (2018): 1104. http://dx.doi.org/10.1182/blood-2018-99-119232.
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Abstract INTRODUCTION: Systemic Mastocytosis (SM) is a rare disease characterized by the clonal proliferation and accumulation of mast cells in the bone marrow, respiratory and gastrointestinal tracts, and organs such as the skin, liver, spleen, and brain. Common symptoms include pruritus, flushing, headache, cognitive impairment, fatigue, diarrhea, abdominal pain, hypotension and skin lesions, as well as an increased risk for osteoporosis and anaphylaxis. SM is currently managed with antihistamines, cromolyn sodium, and leukotriene blocking agents, which lack specificity and efficacy. In addition, glucocorticoids can provide temporary relief in some cases; however long-term treatment with steroids is not appropriate due to their many side effects. Siglec-8 is an inhibitory receptor selectively expressed on human mast cells and eosinophils, and therefore represents a novel target for the treatment of SM. Antibodies to Siglec-8 have been shown to inhibit mast cell activity and induce apoptosis of eosinophils. AK002 is a novel, humanized, non-fucosylated IgG1 monoclonal antibody to Siglec-8. This study evaluates the expression of Siglec-8 and ex vivo activity of AK002 on mast cells and eosinophils in bone marrow biopsies from patients with SM. METHODS: Bone marrow aspirates were obtained from patients clinically diagnosed with SM and processed to remove red blood cells. Multi-color flow cytometry was used to quantify eosinophils and mast cells and to evaluate the activation state of mast cells. The ex vivo activity of AK002 against eosinophils and mast cells was evaluated by flow cytometry. The inhibitory activity of AK002 agaist mast cells was also examined by quantifying cytokine levels in cultured bone marrow aspirate supernatants. RESULTS: All mast cells and eosinophils in bone marrow aspirates from SM patients displayed high Siglec-8 receptor expression (Figure 1). These mast cells also expressed the SM specific markers, CD25 (Figure 1) and CD30 and increased levels of cell surface degranulation markers. The expression of CD25 on mast cells significantly decreased following overnight treatment with AK002. AK002 also significantly reduced the level of mast cell-associated cytokines produced in cultured bone marrow supernatants, including IL-6, IL-8, and TNFα (Figure 2A). These changes in mast cell activity after AK002 treatment were not due to a reduction in mast cell numbers. In contrast, overnight incubation of AK002 significantly reduced the number of bone marrow eosinophils compared to an isotype control (Figure 2B). CONCLUSIONS: Bone marrow aspirates from patients with SM had activated mast cells and eosinophils that displayed robust expression of Siglec-8. AK002 demonstrated SM mast cell inhibition in ex vivo bone marrow aspirates. AK002 also had depleting effects on eosinophils, which may be valuable to SM patients with associated eosinophilia. These encouraging results could represent a novel approach for the treatment of SM. Disclosures Youngblood: Allakos, Inc.: Employment. Brock:Allakos, Inc.: Employment. Leung:Allakos, Inc.: Employment. Chang:Allakos, Inc.: Employment. Bebbington:Allakos, Inc.: Employment. Tomasevic:Allakos, Inc.: Employment.
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Bao, Guangyu, Zhijun Han, Zihe Yan, et al. "Increased Siglec-1 Expression in Monocytes of Patients with Primary Biliary Cirrhosis." Immunological Investigations 39, no. 6 (2010): 645–60. http://dx.doi.org/10.3109/08820139.2010.485625.
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Schadee-Eestermans, Inge L., Elizabeth C. M. Hoefsmit, Marja Van De Ende, Paul R. Crocker, and Timo K. Van Den Berg. "Ultrastructural Localisation of Sialoadhesin (Siglec- 1) on Macrophages in Rodent Lymphoid Tissues." Immunobiology 202, no. 4 (2000): 309–25. http://dx.doi.org/10.1016/s0171-2985(00)80036-4.
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Pillsbury, Claire E., Jairo A. Fonseca, Jodi Dougan, Hasan Abukharma, Linda N. Liu, and Christopher C. Porter. "Siglec-15 Is a Novel Immunomodulatory Protein and Therapeutic Target in Childhood Leukemia." Blood 136, Supplement 1 (2020): 6–7. http://dx.doi.org/10.1182/blood-2020-142833.
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Despite advances that have greatly improved the overall survival of pediatric B cell acute lymphoblastic leukemia (B-ALL), it remains one of the leading causes of cancer-related death in children. Immunotherapy has shown efficacy in treatment of refractory disease, highlighting the need for greater understanding of the immune evasion mechanisms underlying this disease so that additional immune modulating therapeutic strategies can be developed. Siglec-15 (Sig15) was recently reported to have immune modulatory functions in the context of solid tumors. We have found that SIGLEC15 is overexpressed at the RNA level in primary B cell acute lymphoblastic leukemia (B-ALL), acute myelogenous leukemia (AML), and diffuse large B cell lymphoma as compared to healthy donor controls. As compared to healthy donor PBMCs, we have confirmed higher expression of Sig15 at the RNA and protein levels through RT-qPCR, immunoblotting, and flow cytometry across a panel of human B-ALL, AML, DLBCL, and T cell acute lymphoblastic leukemia (T-ALL) cell lines. Knockout of Sig15 expression in a BCR-ABL1+ murine model of B-ALL engrafted in immunocompetent and Rag1-/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure 1). These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies. Additional studies suggest that SIGLEC15 expression is positively regulated by NFκB signaling, which is known to be constitutively activated in certain B-ALL subsets. Importantly, we have observed release of a soluble form of Sig15 (sSig15) from B-ALL cells, which is regulated by PKC and calcineurin-mediated signaling. To discover translational application, we measured sSig15 in the plasma of both healthy and pediatric leukemia patients and found significantly higher levels of sSig15 as compared to healthy individuals (Figure 2; LLD = 5 pg/ml; **P&lt;0.01). Together, these results suggest Siglec-15 is a novel and potent immunosuppressive molecule active in leukemia that may be targeted therapeutically to activate lymphocytes against leukemia cells. Disclosures Abukharma: NextCure, Inc.: Current Employment. Liu:NextCure, Inc.: Current Employment.
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Puryear, Wendy Blay, Hisashi Akiyama, Suzanne D. Geer, et al. "Interferon-Inducible Mechanism of Dendritic Cell-Mediated HIV-1 Dissemination Is Dependent on Siglec-1/CD169." PLoS Pathogens 9, no. 4 (2013): e1003291. http://dx.doi.org/10.1371/journal.ppat.1003291.
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Perez-Zsolt, Daniel, Itziar Erkizia, Maria Pino, et al. "Anti-Siglec-1 antibodies block Ebola viral uptake and decrease cytoplasmic viral entry." Nature Microbiology 4, no. 9 (2019): 1558–70. http://dx.doi.org/10.1038/s41564-019-0453-2.
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Rashmi, Ramachandran, Barrie P. Bode, Ninder Panesar, et al. "Siglec-9 and SHP-1 Are Differentially Expressed in Neonatal and Adult Neutrophils." Pediatric Research 66, no. 3 (2009): 266–71. http://dx.doi.org/10.1203/pdr.0b013e3181b1bc19.
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Grobe, Kay, and Leland D. Powell. "Role of protein kinase C in the phosphorylation of CD33 (Siglec-3) and its effect on lectin activity." Blood 99, no. 9 (2002): 3188–96. http://dx.doi.org/10.1182/blood.v99.9.3188.
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Abstract CD33 (Siglec-3) is a marker of myeloid progenitor cells, mature myeloid cells, and most myeloid leukemias. Although its biologic role remains unknown, it has been demonstrated to function as a sialic acid–specific lectin and a cell adhesion molecule. Many of the Siglecs (including CD33) have been reported to be tyrosine phosphorylated in the cytosolic tails under specific stimulation conditions. Here we report that CD33 is also a serine/threonine phosphoprotein, containing at least 2 sites of serine phosphorylation in its cytoplasmic domain, catalyzed by protein kinase C (PKC). Phosphorylation could be augmented by exposure to the protein kinase–activating cytokines interleukin 3, erythropoietin, or granulocyte-macrophage colony-stimulating factor, in a cytokine-dependent cell line, TF-1. The CD33 cytoplasmic tail was phosphorylated by PKC in vitro, in a Ca++/lipid-dependent manner. CHOK1 cells stably expressing CD33 with cytoplasmic tails of various length also showed phorbol myristate acetate (PMA)-dependent phosphorylation of CD33. Inhibition of CD33 phosphorylation with pharmacologic agents resulted in an increase of sialic acid–dependent rosette formation. Furthermore, the occupancy of the lectin site affected its basal level of phosphorylation. Rosette formation by COS cells expressing a form of CD33 lacking its cytoplasmic domain was not affected by these same agents. These data indicate that CD33 is a phosphoprotein, that its phosphorylation may be controlled by PKC downstream of cytokine stimulation, and that its phosphorylation is cross-regulated with its lectin activity. Notably, although this is the first example of serine/threonine phosphorylation in the subfamily of CD33-like Siglecs, some of the other members also have putative target sites in their cytoplasmic tails.
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Uchil, Pradeep D., Ruoxi Pi, Kelsey A. Haugh, et al. "A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus." Cell Host & Microbe 25, no. 1 (2019): 87–100. http://dx.doi.org/10.1016/j.chom.2018.11.011.
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Bradford, Barry M., Paul R. Crocker, and Neil A. Mabbott. "Peripheral prion disease pathogenesis is unaltered in the absence of sialoadhesin (Siglec-1/CD169)." Immunology 143, no. 1 (2014): 120–29. http://dx.doi.org/10.1111/imm.12294.
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Xiong, Yi-song, Ai-lin Wu, Qiu-shui Lin, et al. "Contribution of monocytes Siglec-1 in stimulating T cells proliferation and activation in atherosclerosis." Atherosclerosis 224, no. 1 (2012): 58–65. http://dx.doi.org/10.1016/j.atherosclerosis.2012.06.063.
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Hurtado-Ziola, Nancy, Justin L. Sonnenburg, and Ajit Varki. "Differential Expression and Function of the CD33-Related Siglecs between Humans and Great Apes." Blood 104, no. 11 (2004): 1466. http://dx.doi.org/10.1182/blood.v104.11.1466.1466.
Full textAbstract:
Abstract The Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are a recently discovered family of mammalian glycan-binding proteins that have been shown to recognize the terminal sialic acids of glycoproteins and glycolipids. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules, which are thought to be primarily expressed on cells of the innate immune system. All CD33rSiglecs are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that usually has two tyrosine-based signaling motifs, one of which conforms to a canonical negative regulatory ITIM motif. Although the true function of the CD33rSiglecs has yet to be discovered, available data are most consistent with an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. CD33rSiglecs also interact with sialic acids on the same cell surface, typically resulting in “masking” of their sialic acid-binding sites. Our recent studies have shown that humans and non-human primates have a similar clustered localization of CD33rSiglec genes, and that true orthologs can generally be identified within each cluster (Angata et al., PNAS, in press). However, humans no longer express CMP-sialic acid hydroxylase (CMAH) the enzyme required to generate one of the potential CD33rSiglec sialic acid ligands called N-glycolylneuraminic acid (Neu5Gc), from its precursor N-acetylneuraminic acid (Neu5Ac). This genetic change occurred after our last common ancestor with the great apes, and dramatically altered the “Sialome” (the sialic acid makeup of a specific species) of humans when compared to that of the great apes. While great ape blood cells express about equal amounts of Neu5Ac and Neu5Gc, human blood cells express almost exclusively Neu5Ac. We also recently discovered that preferential recognition of Neu5Gc is the ancestral condition of most or all of the great ape (chimpanzee and gorilla) CD33rSiglecs (Sonnenburg JL, Altheide TK, Varki A. Glycobiology.14:339–46, 2004). We therefore reasoned that the sudden and major change in the sialome of our hominid ancestors could have had a significant impact on the evolution, binding specificities and expression patterns of CD33rSiglecs. Indeed, we have found that all human CD33rSiglecs can recognize both Neu5Ac and Neu5Gc. This presumably represents an evolutionarily-selected “relaxation” in binding specificity that was necessary to “remask” the Siglecs that had lost their Neu5Gc ligands. Also, there are differences in CD33rSiglec expression on monocytes and neutrophils between humans and great apes (chimp, bonobo, gorilla and orangutan). Furthermore, while great ape cells often show multiple populations with different signal intensities, humans express a single bright peak for each Siglec in flow cytometry. Surprisingly, while humans showed almost no CD33rSiglec expression on lymphocytes, the great apes show a moderate to high expression of some Siglecs on these cells. Total leukocyte expression of some CD33rSiglecs also shows differences between humans and great apes. Overall, CD33rSiglecs appear to be rapidly evolving in primates, with an apparent further acceleration of changes in humans. Additional studies are needed to define the mechanistic details, as well as the implications for human health and disease.