Shoufa Han

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.

High-Affinity Ligand Probes of CD22 Overcome the Threshold Set by cis Ligands to Allow for Binding, Endocytosis, and Killing of B Cells

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Siaα2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear “masked” from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated “cargo” to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.

A self-referenced nanodosimeter for reaction based ratiometric imaging of hypochlorous acid in living cells

Hypochlorous acid (HOCl) is biosynthesized from hydrogen peroxide via catalysis of myeloperoxidase in lysosomes of immunological cells. Despite being harnessed by immune systems against invading pathogens, biogenic HOCl can also damage host tissues and has been associated with a number of diseases. In this edge article, Forster resonance energy transfer based ratiometric imaging of lysosomal HOCl was achieved with silica nanoparticles comprising FITC (donor dye) and a nonfluorescent chemodosimeter which turned into rhodamine (acceptor dye) upon HOCl triggered tandem oxidation and β-elimination of the doped chemodosimeter. The nanodosimeter exhibited distinct biochemical properties relative to small molecule-based free chemodosimeters, e.g. lysosome-homing specificity and compatibility with aqueous media, enabling facile monitoring of lysosomal HOCl by conventional flow cytometry. The nanoprobe would be of broad utility for studies on in vivo generation and the impact of lysosomal HOCl in living cells or even in animals.

Dual colored mesoporous silica nanoparticles with pH activable rhodamine-lactam for ratiometric sensing of lysosomal acidity

Alteration of lysosome acidity has been implicated in many biological events ranging from apoptosis to cancer metastasis, etc. Mesoporous silica nanoparticles doped with acid activable rhodamine-lactam and fluorescein isothiocyanate (FITC) were developed for ratiometric sensing of lysosomal pH changes in live cells with flow cytometry.

Imaging of intracellular acidic compartments with a sensitive rhodamine based fluorogenic pH sensor

The parameters of intracellular acidic compartments are altered in apoptosis, autophagy, cancer metastasis, etc. Low background and selective staining of lysosomes and autophagosomes was achieved with N-(rhodamine 6G)-lactam-ethylenediamine (R6G-EDA) which fluoresces in acidic vesicles via pH mediated ring opening of the intramolecular lactam. Long retention time of R6G-EDA in lysosomes and its exceptional stability against photo-bleaching allow full time tracking of lysosome morphology changes in tumor necrosis factor-α (TNF-α) triggered cell death, suggesting its utility for acidic vesicle studies in cell biology.

Sialoside Analogue Arrays for Rapid Identification of High Affinity Siglec Ligands

The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acalpha2-3Gal and Neu5Acalpha-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function.

Visual and fluorogenic detection of a nerve agent simulant via a Lossen rearrangement of rhodamine-hydroxamate

A visual and fluorogenic detection method for a nerve agent simulant was developed based on a Lossen rearrangement of rhodamine-hydroxamate, in the presence of diethyl chlorophosphate, under alkaline conditions.

Bifunctional CD22 Ligands Use Multimeric Immunoglobulins as Protein Scaffolds in Assembly of Immune Complexes on B Cells

CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells. Surprisingly, anti-NP antibodies of lower valency, IgA (n = 4) and IgG (n = 2), were also found to drive complex formation, though with lower avidity. Ligands bearing alternate linkers of variable length and structure were constructed to establish the importance of a minimal length requirement, and versatility in the structural requirement. We show that the ligand drives assembly of IgM complexes exclusively on the surface of B cells and not other classes of white blood cells that do not express CD22, which lends itself to the possibility of targeting B cells in certain hematopoietic malignancies.

Antigen Delivery to Macrophages Using Liposomal Nanoparticles Targeting Sialoadhesin/CD169

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.

Rhodamine-deoxylactam functionalized poly[styrene-alter-(maleic acid)]s as lysosome activatable probes for intraoperative detection of tumors

Optical probes that could illuminate tumors are promising tools to enable surgical ablation of tumor colonies that are elusive to identify by conventional visual inspection. Polymeric nanoprobes, comprised of a biocompatible poly[styrene-alter-(maleic acid)] carrier and pH responsive rhodamine-deoxylactam, preferentially accumulate in tumors in mice and are activated by lysosomal acidity into highly fluorescent species. The favorable characteristics of passive tumor targeting, e.g. high tumor to normal tissue ratio, long retention time in tumors and low in vivo toxicity, suggest the utility of the nanoprobes in fluorescence guided intraoperative detection of different tumors.