Siglecs (sialic acid immunoglobulin-like lectins) are members of the immunoglobulin gene family that contain sialoside binding N-terminal domains. They are cell surface proteins found predominantly on cells of the immune system. Among them, Siglec-8 is uniquely expressed by human eosinophils and mast cells, as well as basophils. Engaging this structure with antibodies or glycan ligands results in apoptosis in human eosinophils and inhibition of release of preformed and newly generated mediators from human mast cells without affecting their survival. Pro-apoptotic effects are also seen when its closest functional paralog, Siglec-F, on mouse eosinophils is similarly engaged in vitro, and beneficial effects are observed after administration of Siglec-F antibody using models of eosinophilic pulmonary and gastrointestinal inflammation in vivo. Siglec-8 targeting may thus provide a means to specifically inhibit or deplete these cell types. Cell-directed therapies are increasingly sought after by the pharmaceutical industry for their potential to reduce side effects and increase safety. The challenge is to identify suitable targets on the cell type of interest, and selectively deliver a therapeutic agent. By targeting Siglec-8, monoclonal antibodies and glycan ligand-conjugated nanoparticles may be ideally suited for treatment of eosinophil and mast cell-related diseases, such as asthma, chronic rhinosinusitis, chronic urticaria, hypereosinophilic syndromes, mast cell and eosinophil malignancies and eosinophilic gastrointestinal disorders.

Sialoadhesin (Sn, Siglec-1, CD169) is a member of the sialic acid binding Ig-like lectin (siglec) family expressed on macrophages. Its macrophage specific expression makes it an attractive target for delivering antigens to tissue macrophages via Sn-mediated endocytosis. Here we describe a novel approach for delivering antigens to macrophages using liposomal nanoparticles displaying high affinity glycan ligands of Sn. The Sn-targeted liposomes selectively bind to and are internalized by Sn-expressing cells, and accumulate intracellularly over time. Our results show that ligand decorated liposomes are specific for Sn, since they are taken up by bone marrow derived macrophages that are derived from wild type but not Sn−/− mice. Importantly, the Sn-targeted liposomes dramatically enhance the delivery of antigens to macrophages for presentation to and proliferation of antigen-specific T cells. Together, these data provide insights into the potential of cell-specific targeting and delivery of antigens to intracellular organelles of macrophages using Sn-ligand decorated liposomal nanoparticles.

Differentiation of self from non-self is indispensable for maintaining B cell tolerance in peripheral tissues. CD22 and Siglec-G are two inhibitory co-receptors of the BCR that are implicated in maintenance of tolerance to self-antigens. Enforced ligation of CD22 and the BCR by a nanoparticle displaying both antigen and CD22 ligands induces a tolerogenic circuit resulting in apoptosis of the antigen reactive B cell. Whether Siglec-G also has this property has not be investigated in large part due to the lack of a selective Siglec-G ligand. Here, we report the development of a selective high-affinity ligand for Siglec-G and its application as a chemical tool to investigate the tolerogenic potential of Siglec-G. We find that liposomal nanoparticles decorated with antigen and Siglec-G ligand inhibit BCR signaling in both B1 and B2 B cells compared to liposomes displaying antigen alone. Not only is inhibition of B cell activation observed by ligating the BCR with Siglec-G, but robust tolerance towards T-independent and T-dependent antigens is also induced in mice. The ability of Siglec-G to inhibit B cell activation equally in both B1 and B2 subsets is consistent with our observation that Siglec-G is expressed at a relatively constant level throughout numerous B cell subsets. These results suggest that Siglec-G may contribute to maintenance of B cell tolerance towards self-antigens in various B cell compartments.

Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169 + macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169 + macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169 −/− mice and is macrophagedependent, demonstrating that targeting CD169 + macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte-derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells.antigen delivery | immune modulation | antigen presentation

CD33 is an immunomodulatory receptor linked to Alzheimer’s disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo — including aggregated Aβ1–42 — is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.