Siglecs as Immune Cell Checkpoints in Disease.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are expressed on the majority of white blood cells of the immune system and play critical roles in immune cell signaling. Through recognition of sialic acid-containing glycans as ligands, they help the immune system distinguish between self and nonself. Because of their restricted cell type expression and roles as checkpoints in immune cell responses in human diseases such as cancer, asthma, allergy, neurodegeneration, and autoimmune diseases they have gained attention as targets for therapeutic interventions. In this review we describe the Siglec family, its roles in regulation of immune cell signaling, current efforts to define its roles in disease processes, and approaches to target Siglecs for treatment of human disease.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Structural evolution of glycan recognition by a family of potent HIV antibodies.

The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans.

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

Glycan microarrays for decoding the glycome.

In the last decade, glycan microarrays have revolutionized the analysis of the specificity of glycan-binding proteins (GBPs), providing information that simultaneously illuminates the biology mediated by them and decodes the informational content of the glycome. Numerous methods have emerged for arraying glycans in a "chip" format, and glycan libraries have been assembled that address the diversity of the human glycome. Such arrays have been successfully used for analysis of GBPs, which mediate mammalian biology, host-pathogen interactions, and immune recognition of glycans relevant to vaccine production and cancer antigens. This review covers the development of glycan microarrays and applications that have provided insights into the roles of mammalian and microbial GBPs.

Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic ß-Hairpin Structure.

Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans.

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Probing the binding specificities of human Siglecs by cell-based glycan arrays.

Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.

Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers.

Broadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies has been limited. Here, we describe a set of human monoclonal antibodies that define what is, to the best of our knowledge, a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.

Nanoparticles Displaying Allergen and Siglec-8 Ligands Suppress IgE-FcεRI-Mediated Anaphylaxis and Desensitize Mast Cells to Subsequent Antigen Challenge.

Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE-FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow-derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has no protection in mice that do not express Siglec-8. Furthermore, mice protected from anaphylaxis remain desensitized to subsequent allergen challenge because of loss of Ag-specific IgE from the cell surface and accelerated clearance of IgE from the blood. Thus, although expression of human Siglec-8 on murine mast cells does not by itself modulate IgE-FcεRI-mediated cell activation, the enforced recruitment of Siglec-8 to the FcεRI receptor by Sig8L-decorated antigenic liposomes results in inhibition of degranulation and desensitization to subsequent Ag exposure.

Visualization of the HIV-1 Env glycan shield across scales.

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.

Targeting CD22 on memory B cells to induce tolerance to peanut allergens.

BACKGROUND: Circulating IgE and subsequent severe allergic reactions to peanut are sustained and propagated by recall of peanut allergen-specific memory B cells. OBJECTIVES: This study aimed to determine whether targeting mouse and human CD22 on peanut-specific memory B cells induces tolerance to peanut allergens. METHODS: Siglec-engaging tolerance-inducing antigenic liposomes (STALs) codisplaying peanut allergens (Ara h 1, Ara h 2, or Ara h 3) and high-affinity CD22 ligand (CD22L-STALs) were employed in various mouse models (BALB/cJ, C57BL/6, human CD22 transgenic, and NSG) of peanut allergy. To investigate memory B cells, a conferred memory model was used in which splenocytes from peanut-sensitized mice were transferred into naive animals. Reconstituted mice received either CD22L-STALs or an immunogenic liposome control, followed by a peanut allergen boost and later a challenge with individual peanut allergens. To assess the effects of CD22L-STALs on human B cells, PBMCs were injected into NSG mice, followed by administration of human CD22L-STALs (hCD22L-STALs) and later a whole peanut extract boost. Blood was collected to quantify WPE- and Ara h 1-, 2-, and 3-specific immunoglobulins. RESULTS: Mouse CD22L-STALs (mCD22L-STALs) significantly suppressed systemic memory to Ara h 1, Ara h 2, and Ara h 3 in BALB/cJ and C57BL/6 mice, as demonstrated by reduced allergen-specific IgE, IgG1, and anaphylaxis on challenge. Importantly, 2 doses of mCD22L-STALs led to prolonged tolerance for at least 3 months. hCD22L-STALs displayed similar suppression in mice expressing human CD22 on B cells. Finally, human B cells were tolerized in vivo in NSG mice by hCD22L-STALs. CONCLUSIONS: Antigen-specific exploitation of CD22 on memory B cells can induce systemic immune tolerance.

Adaptation of influenza viruses to human airway receptors.

Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential. One natural barrier for transmission of avian IAVs into humans is the specificity of the receptor-binding protein, hemagglutinin (HA), which recognizes sialic-acid-containing glycans on host cells. HAs from human IAVs exhibit "human-type" receptor specificity, binding exclusively to glycans on cells lining the human airway where terminal sialic acids are attached in the α2-6 configuration (NeuAcα2-6Gal). In contrast, HAs from avian viruses exhibit specificity for "avian-type" α2-3-linked (NeuAcα2-3Gal) receptors and thus require adaptive mutations to bind human-type receptors. Since all human IAV pandemics can be traced to avian origins, there remains ever-present concern over emerging IAVs with human-adaptive potential that might lead to the next pandemic. This concern has been brought into focus through emergence of SARS-CoV-2, aligning both scientific and public attention to the threat of novel respiratory viruses from animal sources. In this review, we summarize receptor-binding adaptations underlying the emergence of all prior IAV pandemics in humans, maintenance and evolution of human-type receptor specificity in subsequent seasonal IAVs, and potential for future human-type receptor adaptation in novel avian HAs.

Suppressing Immune Responses Using Siglec Ligand-Decorated Anti-receptor Antibodies.

The sialic acid-binding immunoglobulin-type lectins (Siglecs) are expressed predominantly on white blood cells and participate in immune cell recognition of self. Most Siglecs contain cytoplasmic inhibitory immunoreceptor tyrosine-based inhibitory motifs characteristic of inhibitory checkpoint co-receptors that suppress cell signaling when they are recruited to the immunological synapse of an activating receptor. Antibodies to activatory receptors typically activate immune cells by ligating the receptors on the cell surface. Here, we report that the conjugation of high affinity ligands of Siglecs to antibodies targeting activatory immune receptors can suppress receptor-mediated activation of immune cells. Indeed, B-cell activation by antibodies to the B-cell receptor IgD is dramatically suppressed by conjugation of anti-IgD with high affinity ligands of a B-cell Siglec CD22/Siglec-2. Similarly, degranulation of mast cells induced by antibodies to IgE, which ligate the IgE/FcεR1 receptor complex, is suppressed by conjugation of anti-IgE to high affinity ligands of a mast cell Siglec, CD33/Siglec-3 (CD33L). Moreover, the anti-IgE-CD33L suppresses anti-IgE-mediated systemic anaphylaxis of sensitized humanized mice and prevents anaphylaxis upon subsequent challenge with anti-IgE. The results demonstrate that attachment of ligands of inhibitory Siglecs to anti-receptor antibodies can suppress the activation of immune cells and modulate unwanted immune responses.

DeGlyPHER: An Ultrasensitive Method for the Analysis of Viral Spike N-Glycoforms.

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single-pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for the analysis of site-specific glycosylation of any glycoprotein.

Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection.

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans.IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

Migration-based selections of antibodies that convert bone marrow into trafficking microglia-like cells that reduce brain amyloid ß.

One goal of regenerative medicine is to repair damaged tissue. This requires not only generating new cells of the proper phenotype, but also selecting for those that properly integrate into sites of injury. In our laboratory we are using a cell-migration-based in vivo selection system to generate antibodies that induce cells to both differentiate and selectively localize to different tissues. Here we describe an antibody that induces bone marrow stem cells to differentiate into microglia-like cells that traffic to the brain where they organize into typical networks. Interestingly, in the APP/PS1 Alzheimer's disease mouse model, these induced microglia-like cells are found at sites of plaque formation and significantly reduce their number. These results raise the intriguing question as to whether one can use such antibody-induced differentiation of stem cells to essentially recapitulate embryogenesis in adults to discover cells that can regenerate damaged organ systems.