Binding Immunoglobulin-like Lectins Research Articles

A Siglec3-like lectin from Nile tilapia (Oreochromis niloticus): Transcriptional profiling upon Streptococcus agalactiae infection and its potential role in immune escape

Siglec-3, also known as CD33, is a type of sialic acid binding immunoglobulin-like lectin that plays a role in regulating cellular activation in the innate immune system and subsequent inflammatory responses. In this study, we cloned the cDNA encoding OnSiglec3-like ORF sequence (1170 bp) in Nile tilapia, contained two Ig-like domains in the extracellular region and one inhibitory ITIM-like motif in the intracellular region. Through qRT-PCR analysis, we found that the mRNA of OnSiglec3-like lectin was highly expressed in the brain and head kidney. When challenged with Streptococcus agalactiae both in vivo and in vitro, we observed a significant up-regulation in the expression of OnSiglec3-like lectin in the head kidney tissues and leukocytes, respectively. Additionally, we investigated the interaction between a recombinant extracellular region protein (referred to as (r)exSiglec3-like protein) and three different phenotypes of S. agalactiae: WT (wild type), ∆cps (cps-deficient mutant), and ∆neuA (sia-deficient mutant) using ELISA. We found that the exOnSiglec3-like protein, when combined with RNA interference and bacterial infection, significantly enhanced the phagocytic ability against S. agalactiae. Furthermore, the expression of four common inflammatory factors (OnIL-6, OnIL-10, OnTNF-α, and OnTGF-β) showed a significant up-regulation. Overall, this study suggests that OnSigelc3-like lectin may play a role in host defence against bacterial infection and mediate immune escape in Nile tilapia.

The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

Outcomes for patients with relapsed or refractory (r/r) hematologic malignancies remain dismal despite therapeutic advances over the last several years. This is particularly striking in patients with r/r aggressive mature B-cell lymphomas such as Diffuse Large B-cell Lymphoma (DLBCL) and Burkitt's Lymphoma (BL). With use of intensive multiagent chemo-immunotherapy, DLBCL, which accounts for approximately 10-20% of pediatric Non-Hodgkin's Lymphoma (NHL) cases and is the most common subtype in adults, is highly curable with 5-year event free survival >75%. However, approximately 30-40% of adults diagnosed with DLBCL will become refractory to or relapse after first-line treatment. Yet even with intensive salvage regimens, r/r DLBCL remains a clinical challenge. There remains an unmet need to identify specific defects in the immune response, among other pathways, that drive lymphomagenesis that will in turn inform the development of novel therapeutics to improve survival in this population of patients. Siglec-15 (Sig-15), a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, recent reports have demonstrated inhibition of Sig-15, has a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies and acute leukemias, a role for Siglec-15 in promoting disease progression in aggressive B-cell lymphomas has not yet been described. We have found higher expression of SIGLEC15 in DLBCL cell lines compared to normal B cells at the RNA level, through analyses of the publicly available ONCOMINE database. This was confirmed at the protein level by western blot demonstrating higher Sig-15 expression in various lymphoma cell lines (RAJI, SUDHL6, OCI-LY10, HBL-1) compared to healthy donor PBMCs. Further, immunohistochemistry evaluating Sig-15 expression was performed on a tumor microarray consisting of 139 cases of DLBCL from adult patients along with validation samples from 15 primary pediatric lymphoma cases (DLBCL, BL and Anaplastic Large Cell Lymphoma (ALCL)). Sig-15 was found to be highly expressed, with histologic scores ranging from weak (1+) to strong (3+) in 124 out of the 139 adult cases (Figure 1A) and in all pediatric lymphoma samples but with distinct staining patterns observed in the aggressive B-cell lymphomas. Specifically, Sig-15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt's lymphoma. Expression is more variable in ALCL, primarily elevated in the cytoplasm at low levels and/or in macrophages (Figure 1A). There is heterogeneity in Sig-15 expression with preliminary analysis of RNA-seq data from a cohort of 693 DLBCL patient samples demonstrates higher levels in the germinal center B-cell (GCB) versus activated B-cell molecular (ABC) subtype of DLBCL when compared to healthy controls (Kruskal-Wallis, *p = 0.041). Finally, using a well-established murine lymphoma model, A20 cells were stably transduced with control, non-silencing shRNA (shNS) or shRNA against Sig-15 and injected into un-irradiated immune competent (wild type, WT) or immune deficient ( Rag1 -/-) BALB/C mice and monitored for signs of lymphoma development. Knockdown of Sig-15 in A20 cells abrogates disease progression in WT but not immune deficient mice (Log-rank, **p <0.0001, n=10 mice/group), consistent with a role for Sig-15 in immune evasion in lymphoma (Figure 1B). Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells.

The Contribution of Serum Sialic Acid Binding Immunoglobulin-Like Lectin 1(sSIGLEC-1) as an IFN I Signature Biomarker in the Progression of Atherosclerosis in Egyptian Systemic Lupus Erythematosus (SLE) Patients.

Clinical symptoms of systemic lupus erythematosus (SLE), such as atherosclerosis and related cardiovascular diseases, are caused by inflammatory cytokines and endothelial cell damage. The serum sialic acid binding immunoglobulin-like lectin 1 (sSIGLEC-1) is thought to be an alternative biomarker of IFN signature and may have a role in the pathogenesis of atherosclerosis. The aim of the study was to measure the levels of sSIGLEC-1 in the serum of SLE patients in comparison to a control group and examine the associations between sSIGLEC-1, SLEDAI, lipid profile, oxidized low density lipoprotein (oxLDL), and carotid intima media thickness (CIMT) to investigate whether sSIGLEC-1 participates in the development of atherosclerosis. sSIGLEC-1 levels were tested in 53 patients and 20 volunteers using ELISA kit. Duplex measurements were performed on all subjects to measure CIMT. SLE patients had significantly higher values of sSIGLEC-1 (P < 0.0001), total cholesterol (P = 0.029), triglycerides (P = 0.001), low density lipoprotein (P = 0.032), oxLDL (P = 0.001), right CIMT (P = 0.0099) and a significantly lower value of high-density lipoprotein (P = 0.04) when compared to controls. sSIGLEC-1 had significant positive correlations with right CIMT (r = 0.5, P < 0.0002) and oxLDL (r = 0.67, P < 0.0001) in all SLE patients. When compared to non-dyslipidemic patients, the dyslipidemic group exhibited significantly higher levels of all previous parameters except HDL and left CIMT. Circulating form of SIGLEC-1 accelerates atherosclerosis and provides a simple way to predict the occurrence of atherosclerosis in SLE patients.

Association of Sialic Acid–Binding Immunoglobulin-Like Lectin 15 With Phenotypes in Esophageal Squamous Cell Carcinoma in the Setting of Neoadjuvant Chemoradiotherapy

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a novel immune checkpoint molecule that is highly homologous to programmed cell death ligand 1 (PD-L1), but information remains limited about its role in esophageal squamous cell carcinoma (ESCC). To explore the expression pattern and association of Siglec-15 with outcomes among patients with ESCC who received neoadjuvant chemoradiotherapy (CRT). This retrospective cohort study was conducted at an academic institution in China. Participants included patients with ESCC who underwent neoadjuvant CRT and esophagectomy between June 2002 and December 2018. Multiplexed immunofluorescence staining was used to evaluate the expression of Siglec-15 and PD-L1 in tumor cells (TCs) or tumor-associated macrophages based on pre-CRT biopsies. Different immune phenotypes have been proposed and further validated in an independent cohort. Data analysis was conducted from January to May 2021. Siglec-15 or PD-L1 positivity vs negativity. Pathologic complete response (pCR), overall survival (OS), and recurrence-free survival (RFS). Of 130 participants (median [range] age, 56 [42-73] years; 108 [83.1%] male participants) in the primary cohort, 58 patients (44.6%) achieved a pCR after neoadjuvant CRT. Siglec-15 and PD-L1 were detected in both TCs and macrophages. The percentage of Siglec-15-positive macrophages was notably higher than that of Siglec-15-positive TCs (median [IQR]: 34.4% [12.7%-64.3%] vs 4.8% [0.7%-25.6%]; P < .001). TC-Siglec-15 expression was significantly and positively associated with macrophage-Siglec-15 expression (r = 0.78; P < .001). Siglec-15 positivity was significantly associated with a higher rate of pCR (37 of 70 [52.9%] vs 21 of 60 [35.0%]; P = .04), more favorable OS (hazard ratio [HR], 0.46; 95% CI, 0.25-0.85; P = .01), and RFS (HR, 0.48; 95% CI, 0.26-0.88; P = .02). However, PD-L1 positivity in TCs was negatively associated with survival. Stratification analysis further revealed that patients with combined Siglec-15 positivity and PD-L1 negativity had better survival than those with other phenotypes. Major findings were reproducible in a validation cohort with 55 patients. In this cohort study of patients with ESCC receiving neoadjuvant CRT, Siglec-15 positivity was associated with a better pathological response and more favorable survival. Siglec-15 could serve as a novel biomarker to identify potential candidates that may benefit from immunotherapy combined with CRT.

Tailored Multivalent Targeting of Siglecs with Photosensitizing Liposome Nanocarriers.

The modification of surfaces with multiple ligands allows the formation of platforms for the study of multivalency in diverse processes. Herein we use this approach for the implementation of a photosensitizer (PS)‐nanocarrier system that binds efficiently to siglec‐10, a member of the CD33 family of siglecs (sialic acid (SA)‐binding immunoglobulin‐like lectins). In particular, a zinc phthalocyanine derivative bearing three SA moieties (PcSA) has been incorporated in the membrane of small unilamellar vesicles (SUVs), retaining its photophysical properties upon insertion into the SUV's membrane. The interaction of these biohybrid systems with human siglec‐10‐displaying supported lipid bilayers (SLBs) has shown the occurrence of weakly multivalent, superselective interactions between vesicle and SLB. The SLB therefore acts as an excellent cell membrane mimic, while the binding with PS‐loaded SUVs shows the potential for targeting siglec‐expressing cells with photosensitizing nanocarriers.

Abstract 3845: Elucidating the role and mechanisms by which siglec-15 promotes immune dysregulation in lymphoma

Abstract Despite important advances in therapeutic options accounting for improved outcomes in childhood cancers, there remains a significant subset of patients with hematologic malignancies for whom outcomes remain dismal. Notably among this group are patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). DLBCL accounts for approximately 10-20% of pediatric Non-Hodgkin’s Lymphoma (NHL) cases and with intensive multiagent chemotherapy the 5-year event free survival is ~90%. However, for those with progressive or relapsed disease, survival is dismal at &amp;lt;30% and remains a clinical challenge even with use of intensive salvage regimens. As such, identifying novel agents that will increase survival in this group highlights an unmet need. Siglec-15, a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, inhibiting Siglec-15, either through genetic knockout or knockdown, had a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies, a role for Siglec-15 in promoting disease progression in hematologic malignancies has not yet been described. We have evaluated Siglec-15 expression in primary human lymphoma patient samples as well as various lymphoma (human and mouse) cell lines using western blot, quantitative PCR as well immunohistochemistry and immunofluorescence methods. We are evaluating the effect of inhibiting expression of Siglec-15 through genetic downregulation in human lymphoma cell lines as well as the well-established murine lymphoma cell line A20 on T cell function and proliferation using methods such as flow cytometry and ELISA. Analyses of public RNA-seq data sets demonstrates higher Sig15 expression in DLBCL cells compared to normal B cells. Western blot shows higher Sig15 expression in lymphoma cell lines compared to PBMC. IHC of a tumor microarray and validation samples from children shows high Sig15 expression in NHL samples with distinct staining patterns based on subtype. Specifically, Sig15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt’s lymphoma, with more variable expression in anaplastic large cell lymphoma, primarily in the cytoplasm at low levels and/or in cells with morphology consistent with macrophages. In addition, preliminary data suggest more than one isoform of Sig15, raising the possibility of alternative functions. Lastly, knockdown of Sig15 in A20 cells abrogates disease progression in immune competent but not immune deficient recipients, consistent with a role for Sig15 in immune evasion in lymphoma. Together, these data implicate Sig15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells. Citation Format: Dailia B. Francis, Jodi Dougan, Claire Pillsbury, Sunita Park, Linda Liu, Christopher Porter. Elucidating the role and mechanisms by which siglec-15 promotes immune dysregulation in lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3845.

Siglec Ligands.

A dense and diverse array of glycans on glycoproteins and glycolipids decorate all cell surfaces. In vertebrates, many of these carry sialic acid, in a variety of linkages and glycan contexts, as their outermost sugar moiety. Among their functions, glycans engage complementary glycan binding proteins (lectins) to regulate cell physiology. Among the glycan binding proteins are the Siglecs, sialic acid binding immunoglobulin-like lectins. In humans, there are 14 Siglecs, most of which are expressed on overlapping subsets of immune system cells. Each Siglec engages distinct, endogenous sialylated glycans that initiate signaling programs and regulate cellular responses. Here, we explore the emerging science of Siglec ligands, including endogenous sialoglycoproteins and glycolipids and synthetic sialomimetics. Knowledge in this field promises to reveal new molecular pathways controlling cell physiology and new opportunities for therapeutic intervention.

Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells

Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counter-receptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7. We then confirmed the counter-receptor for Siglec-7 as leukosialin (CD43) through mass spectrometry, immunoprecipitation, and proximity labeling. Additionally, we demonstrated that the cytotoxicity of NK cells toward K562 cells was suppressed by overexpression of leukosialin in a Siglec-7-dependent manner. Taken together, our data suggest that leukosialin on K562 is a counter-receptor for Siglec-7 on NK cells and that a cluster of the Sia-containing glycan epitope on leukosialin is key as trans-ligand for unmasking the cis-ligand.

The conserved arginine residue in all siglecs is essential for Siglec-7 binding to sialic acid

Siglecs are sialic acid (Sia)-binding immunoglobulin-like lectins; the majority of Siglecs functions as transmembrane receptors on the immune cells via Sia residues. Recently, a new Sia binding site in Siglec-7, termed site 2, where arginine (R) 67 was critical, was identified by computational modeling and biochemical analyses, relative to the primary Sia binding site, termed site 1, containing critical R124. Here, the presence of a new essential R94 residue, which is completely conserved among all identified Siglecs, was demonstrated. A mutation of R94 residue in Siglec-7 led to the disappearance of the Sia binding property, similar to a site 1 mutation (R124A). R94 is close to R67 in site 2, and site 2 mutations at either of them abolished the ligand-binding properties to both gangliosides and glycoproteins. These data suggest that, in addition to site 1, the conserved R residue among Siglecs in site 2 is another functional site.

Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model.

In order to combat invading pathogens neutrophils can release neutrophil extracellular traps (NETs). However, since NETs can also damage endogenous cells, several control mechanisms for the formation of NETs must work effectively. For instance, neutrophil activation is silenced within blood circulation by the binding of sialylated glycoconjugates to sialic acid binding immunoglobulin-like lectins (Siglecs) on neutrophils. As neutrophils are recruited within the female reproductive tract, after mating, a comparable mechanism may also take place within the bovine cervix to prevent an exaggerated NET formation and thus, infertility. We examined, if the highly glycosylated mucins, which are the major functional fraction of biomolecules in mucus, represent a potential regulator of NET formation. The qPCR data revealed that in polymorphonuclear neutrophils (PMNs) inhibitory Siglecs are the most frequently expressed Siglecs and might be a potential target of sialylated glycans to modulate the activation of PMNs. Remarkably, the addition of bovine cervical mucins significantly inhibited the formation of NET, which had been induced in response to lipopolysaccharides (LPS) or a combination of phorbol myristate acetate (PMA) and ionomycin. The inhibitory effects were independent of the stage of estrous cycle (estrus, luteal, and follicular mucins). PMNs retained their segmented nuclei and membrane perforation was prevented. However, the inhibitory effects were diminished, when sialic acids were released under acidic conditions. Comparable results were achieved, when sialic acids were targeted by neuraminidase digestion, indicating a sialic acid dependent inhibition of NET release. Thus, bovine cervical mucins have an anti-inflammatory capability to modulate NET formation and might be further immunomodulatory biomolecules that support fertility.

A Cartography of Siglecs and Sialyltransferases in Gynecologic Malignancies: Is There a Road Towards a Sweet Future?

Altered surface glycosylation is a key feature of cancers, including gynecologic malignancies. Hypersialylation, the overexpression of sialic acid, is known to promote tumor progression and to dampen antitumor responses by mechanisms that also involve sialic acid binding immunoglobulin-like lectins (Siglecs), inhibitory immune receptors. Here, we discuss the expression patterns of Siglecs and sialyltransferases (STs) in gynecologic cancers, including breast, ovarian, and uterine malignancies, based on evidence from The Cancer Genome Atlas. The balance between sialosides generated by specific STs within the tumor microenvironment and Siglecs on leukocytes may play a decisive role for antitumor immunity. An interdisciplinary effort is required to decipher the characteristics and biological impact of the altered tumor sialome in gynecologic cancers and to exploit this knowledge to the clinical benefit of patients.

Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor

Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)-like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors.

Steering Siglec-Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry.

Sialic acid sugars that terminate cell-surface glycans form the ligands for the sialic acid binding immunoglobulin-like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross-reactivity and led to the discovery of three selective Siglec-5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec-3 dampened the activation of Siglec-3+ monocytic cells through the NF-κB and IRF pathways.

Olig2 is expressed late in human eosinophil development and controls Siglec-8 expression.

Oligodendrocyte transcription factor 2, a basic helix-loop-helix transcription factor that binds to E-box motifs, is known to have a key role in determining lineage specification of oligodendrocytes and motor neurons. In the present study, we report that oligodendrocyte transcription factor 2 is expressed in human eosinophils and involved in transcriptional activation of the gene encoding sialic acid binding immunoglobulin-like lectin 8 (Siglec-8), a late eosinophil-differentiation marker known to exert eosinophil apoptosis. When cord blood CD34+ hematopoietic stem cells differentiated toward eosinophils during a 24-d culture period, oligodendrocyte transcription factor 2 protein was expressed in cord blood eosinophils on d 24, a time when cord blood eosinophils are considered fully differentiated, whereas it was not detectable on d 18 or at earlier time points. Oligodendrocyte transcription factor 2 protein was also abundantly expressed in human peripheral-blood eosinophils but not in neutrophils, monocytes, lymphocytes, or cord blood mast cells. RNA sequencing analysis showed that numerous genes, especially those encoding eosinophil surface molecules, were highly up-regulated along with OLIG2 Among the genes examined, SIGLEC-8 messenger RNA and protein were markedly down-regulated in parallel with OLIG2 by an oligodendrocyte transcription factor 2 small interfering RNA or a short hairpin RNA, as evidenced by real-time polymerase chain reaction, fluorescence-activated cell sorting, and Western blot analyses. In reporter gene and chromatin immunoprecipitation experiments, an E-box in the first intron was found to stimulate SIGLEC-8 gene transcription and to bind oligodendrocyte transcription factor 2. Hence, at least one important aspect of eosinophil differentiation is regulated by oligodendrocyte transcription factor 2, a transcription factor that has not previously been reported, to our knowledge, in normal granulocytes.

Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion

Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection.

Lectin Galactoside-Binding Soluble 3 Binding Protein (LGALS3BP) is a Cancer-Associated Ligand for Inhibitory Siglecs

ABSTRACT Tumor cells subvert the control of the immune system by downregulation of their antigenicity and production of an immunosuppressive microenvironment including the upregulation and engagement of inhibitory receptors on immune cells. Therapeutic strategies have demonstrated that the immune system can be reactivated and control established cancers by blocking inhibitory receptors on immune cells such CTLA-4 and PD1. While such activation of the immune system is successful in some patients, many patients still show cancer progression after some time. Thus, the definition of new targetable immunomodulatory pathways is needed to improve the outcome in those patients. Recent evidence suggests that sialic acid dependent ligands on tumor cells can engage inhibitory sialic acid binding immunoglobulin-like lectins (Siglecs) on NK cells and cells of the myelomonocytic lineage and thereby facilitate evasion of immune-mediated killing. Moreover, the presence of a natural variant of Siglec-9 with reduced binding capacity to sialic acid dependent ligands in patients with non-small cell lung cancer improved the two year survival in a retrospective multivariate analysis. Here we identify a novel cancer-associated ligand for immuno-inhibitory Siglecs by affinity chromatography and subsequent proteomic analysis. LectinGalactoside-Binding Soluble 3 Binding Protein (LGALS3BP) bound to various inhibitory Siglecs including Siglec-5, Siglec-9 and Siglec-10 with high affinity. LGALS3BP was previously found to be upregulated in various carcinomas such as breast, colorectal, prostate and lung cancer and linked to advanced stage and poor prognosis. The exact function during cancer progression, however, was not yet defined. Our findings provide a novel insight into how LGALS3BP could promote immune evasion by inhibiting immune cell activation through engagement of Siglecs and defines LGALS3BP-Siglec interactions as potential novel target to interfere with cancer progression and reactivate the immune system against carcinomas. Disclosure All authors have declared no conflicts of interest.

Interaction of Streptococcus agalactiae and Cellular Innate Immunity in Colonization and Disease.

Streptococcus agalactiae (Group B streptococcus, GBS) is highly adapted to humans, where it is a normal constituent of the intestinal and vaginal flora. Yet, GBS has highly invasive potential and causes excessive inflammation, sepsis, and death at the beginning of life, in the elderly and in diabetic patients. Thus, GBS is a model pathobiont that thrives in the healthy host, but has not lost its potential virulence during coevolution with mankind. It remains incompletely understood how the innate immune system contains GBS in the natural niches, the intestinal and genital tracts, and which molecular events underlie breakdown of mucocutaneous resistance. Newborn infants between days 7 and 90 of life are at risk of a particularly striking sepsis manifestation (late-onset disease), where the transition from colonization to invasion and dissemination, and thus from health to severe sepsis is typically fulminant and not predictable. The great majority of late-onset sepsis cases are caused by one clone, GBS ST17, which expresses HvgA as a signature virulence factor and adhesin. In mice, HvgA promotes the crossing of both the mucosal and the blood–brain barrier. Expression levels of HvgA and other GBS virulence factors, such as pili and toxins, are regulated by the upstream two-component control system CovR/S. This in turn is modulated by acidic epithelial pH, high glucose levels, and during the passage through the mouse intestine. After invasion, GBS has the ability to subvert innate immunity by mechanisms like glycerinaldehyde-3-phosphate-dehydrogenase-dependent induction of IL-10 and β-protein binding to the inhibitory phagocyte receptors sialic acid binding immunoglobulin-like lectin 5 and 14. On the host side, sensing of GBS nucleic acids and lipopeptides by both Toll-like receptors and the inflammasome appears to be critical for host resistance against GBS. Yet, comprehensive models on the interplay between GBS and human immune cells at the colonizing site are just emerging.

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

C-4 Modified Sialosides Enhance Binding to Siglec-2 (CD22): Towards Potent Siglec Inhibitors for Immunoglycotherapy

The regulatory functions of Siglecs (sialic acid binding immunoglobulin-like lectins) in the immune system provide opportunities for innovative therapeutic strategies for a wide range of immunological disorders or cancer (immunoglycotherapy). Siglec-2 (CD22), as a consequence of its pivotal role in B cell activation, has become an attractive target for therapies of autoimmune diseases and B cell-derived nonHodgkin s lymphoma (NHL). NHL is among the ten most common cancers with over 20000 deaths in 2010 for the US alone. Siglec-2 binds with high preference to a(2,6)-linked sialic acids (Sia), such as Neu5Aca(2,6)lactosamine (Scheme 1). Neu5Aca2Me (1) interacts with Siglec-2 mainly through 1) the negative charge on its carboxylate group, 2) the C-5 N-acetamido substituent, and 3) the glycerol side chain. Furthermore, replacement of the C-9 hydroxy group by an amino group did not interfere with binding to Siglecs. Crystallographic studies on Siglec-1 (sialoadhesin, Sn) demonstrated that acylation of this amino group enhances the overall affinity of the ligand for Siglecs by two to three orders of magnitude. The first breakthrough in the development of potent Siglec-2 inhibitors was the design of 9biphenylcarboxamido Neu5Aca2Me (9-BPC-Neu5Aca2Me, 2) which has a more than two orders of magnitude higher affinity to Siglec-2 than 1, and 2 has demonstrated potential to modulate signal transduction in B cells. Furthermore, based on 2, compounds were developed, which kill B cell lymphoma cells. Structural studies and modifications of the C-5 N-acyl substituent and the C-2 aglycon moiety of Nacetylneuraminic acid (Neu5Ac) have led to further improvement in affinity. Herein we report, for the first time the design, synthesis, and evaluation of a novel class of disubstituted Neu5Ac derivatives that is modified at the C-4 and C-9 positions of 1. Our structure-based design approach resulted in a promising novel lead compound 9-biphenylcarboxamido-4-m-nitrophenylcarboxamido-4,9-dideoxy Neu5Aca2Me (9-BPC-4mNPC-Neu5Aca2Me, 6b) that has sub-micromolar affinity for Siglec-2 and may provide a pathway for immunoglycotherapy strategies. An evaluation of our homology model (see Supporting Information) for Siglec-2 and other Siglecs led us to hypothesize that substituents at C-4 may provide additional interactions. To address this hypothesis we posed the following questions: 1) Can C-4 substituents enhance the interaction with Siglecs? 2) Do they interact specifically with the protein? 3) Do C-4 and C-9 modifications act synergistically? 4) Do the C-4 modified Neu5Ac derivatives bind to the same binding site as other Sia, such as 1?

SIGLEC1 and SIGLEC7 expression in circulating monocytes of patients with multiple sclerosis

Background: Sialic acid binding immunoglobulin-like lectins (Siglecs) are cell surface receptors that recognize sialic acids and may attenuate immune responses and reduce inflammation. Objective: The purpose of this study was to investigate the role of two members of the Siglec family, SIGLEC1 and SIGLEC7, in the clinical course and disease activity of patients with multiple sclerosis (MS). Methods: SIGLEC1 and SIGLEC7 expression was determined by flow cytometry in the blood monocytes of 16 healthy controls and 55 untreated MS patients (13 primary progressive MS (PPMS) patients, 13 secondary progressive MS (SPMS) patients and 29 relapsing–remitting MS (RRMS) patients (18 during clinical remission and 11 during relapse)). Results: SIGLEC1 expression by CD14+ monocytes was significantly increased in MS patients compared with controls (p=0.025 for percentage of positive cells; p=0.007 for mean fluorescence intensity (MFI)). Stratification of patients into different clinical forms revealed increased SIGLEC1 expression in patients with progressive forms of the disease, particularly in those with PPMS (p=0.003 for percentage of positive cells and p=0.001 for MFI when compared with controls; p=0.031 for percentage of positive cells when compared with RRMS patients). Both inflammatory and resident monocytes contributed to the increase in SIGLEC1 expression observed in PPMS patients. SIGLEC7 expression was significantly up-regulated in blood monocytes from RRMS during relapse compared with patients during clinical remission (p=0.001 for MFI). Conclusions: These findings suggest roles for SIGLEC1 in the chronic progressive phases of MS and for SIGLEC7 in acute disease activity.