Activating B Cell Signaling with Defined Multivalent Ligands

Puffer, E; Pontrello, Jason K.; Hollenbeck, Jessica J.; Kink, John A.; Kiessling, Laura L.

doi:10.1021/cb600489g

Cited by 163 publications

(216 citation statements)

References 76 publications

(132 reference statements)

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“…Both parameters were chosen to elicit robust BCR signaling. Specifically, the hapten density for both homopolymers and copolymers was 33-36%, and we had shown previously that this level of substitution results in BCR binding and signaling (29). The level of substitution of CD22L was 25%, which we anticipated would be sufficient to allow for CD22 binding.…”

Section: Resultsmentioning

confidence: 90%

“…Our strategy to illuminate CD22 function depends upon the design and synthesis of chemical probes. We and others had shown previously that polymeric antigens can induce B-cell activation (28,29). We reasoned that polymeric antigens that display a BCR-binding epitope and a CD22 ligand should provide an excellent test for trans interactions in CD22 signaling.…”

Section: Resultsmentioning

confidence: 98%

“…Antigenic polymers were prepared as previously described (29). Briefly, a succinimidyl ester-substituted norbornene monomer was dissolved in a deoxygenated solution of dichloromethane to which a ruthenium carbene initiator was added.…”

Section: Methodsmentioning

confidence: 99%

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Sialylated multivalent antigens engage CD22in transand inhibit B cell activation

Courtney¹,

Puffer²,

Pontrello

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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121

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CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.B cell antigen receptor ͉ multivalency ͉ sialic acid ͉ siglec ͉ autoimmunity T he initiation of an immune response or the prevention of autoimmunity depends upon the ability of the B cell antigen receptor (BCR) to transmit signals that positively or negatively regulate B lymphocyte survival, proliferation, and differentiation (1). To avoid detrimental autoimmune responses, a means of differentiating between foreign and self-antigens is required; coreceptors that modulate BCR signaling can ensure that these distinctions are made. CD22 is an inhibitory coreceptor that can attenuate BCR signaling (2, 3). CD22 null mice possess hyperresponsive B cells (4), illustrating a role for CD22 in establishing a threshold for B cell activation. Specifically, an increase in intracellular Ca 2ϩ ion concentration is a hallmark of B cell activation (5, 6), and B cells isolated from CD22 null mice display increased Ca 2ϩ flux in response to antigen (4, 7). Thus, loss of CD22 results in a lowering of the threshold for B cell activation. Other data also support this conclusion: CD22 null mice exhibit increased serum IgM concentrations, decreased surface IgM levels on peripheral B cells, increased induction of apoptosis in response to BCR crosslinking, and increased serum autoantibody titers (8). These observations are consistent with the loss of CD22 leading to increased sensitivity and chronic B cell activation.The process of B cell activation ensues upon binding of multivalent antigen to the BCR. Antigen-induced clustering elicits phosphorylation of the cytoplasmic immunoreceptor tyrosinebased activation motifs (ITAMs), which are present in the BCRassociated signaling proteins Ig␣/␤. The phosphorylation reaction is catalyzed by Src-family kinases such as Lyn. Upon phosphorylation of the BCR components, Syk kinase is recruited to the BCR signaling complex (9). Syk is essential for propagating BCR signaling (10, 11). It acts along with...

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 98%

See 1 more Smart Citation

Sialylated multivalent antigens engage CD22in transand inhibit B cell activation

Courtney¹,

Puffer²,

Pontrello

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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121

178

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“…We had shown previously that DNP-substituted polymers promote B cell receptor internalization. 9 The labeled polymers reveal that the B cell receptor and polymer 9b (M:I=500) Figure 2E). In contrast, polymers lacking DNP groups were not taken up (see the Supporting Information).…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 98%

“…We had previously used ROMP to synthesize multivalent ligands that bear antigenic dinitrophenyl (DNP) epitopes, which bind to a cell line expressing a DNP-specific B cell receptor. 9 Thus, we modified the pre-existing synthetic route to generate DNP-substituted polymers bearing a terminal azide group. A norbornene derivative that bears a succinimidyl ester was polymerized using a ruthenium initiator 21 to generate polymers of defined lengths (Scheme 2).…”

mentioning

confidence: 99%

Synthesis of Fluorogenic Polymers for Visualizing Cellular Internalization

2008

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The binding of a polymeric ligand to a cell surface receptor can promote its internalization. Methods to track and visualize multivalent ligands within a cell can give rise to new therapeutic strategies and illuminate signaling processes. We have used the features of the ring-opening metathesis polymerization (ROMP) to develop a general strategy for synthesizing multivalent ligands equipped with a latent fluorophore. The utility of ligands of this type is highlighted by visualizing multivalent antigen internalization in live B cells.Advances in organic and polymer chemistry are providing access to new classes of bioactive polymers. 1 With the ability to generate synthetic macromolecules that vary in structure and physical properties, the applications of polymers have expanded rapidly. Some uses include as substrates for cell growth and differentiation, 2 vehicles for drug delivery, 3 therapeutics, 4 and molecules for studying cell signaling. 5 For some biological applications, bioactive polymers must be taken up into cells. Still, little is known about the cellular internalization and trafficking of polymers. Here, we present an approach for following these processes.The bioactive polymers that we focused upon were those generated by ruthenium carbeneinitiated ring-opening metathesis polymerization (ROMP). It was shown that ROMP could be used to generate biologically-active polymers over 10 years ago. 6 Since that time, ROMP has been used produce materials with a variety of biological activities. 7 Continuous advances in catalyst development and polymerization techniques have provided methods to control key features of polymer structure, including their length and the functional groups they present. 8 Ruthenium carbene initiators tolerate a diverse array of functional groups; therefore, they can be used synthesize highly functionalized polymers with tailored biological activities. ROMP initially was used to generate polymers that act on the outside of the cell, but more recently, it has been employed to assemble compounds that can be internalized by cells. Our interest in polymer uptake was prompted by our studies of B cell signaling. 9 For example, polymers generated by ROMP that display antigenic epitopes can promote antibody production in vivo. The ability of these polymers to activate this process depends upon their interactions with the antigen-specific B cell receptor (BCR), a membrane-bound immunoglobulin on the surface of B cells. These polymers bind to the specific B cell receptor to activate signaling but they also promote its internalization. We therefore anticipated that, like other antigens, these polymers would be taken up by endocytosis. To investigate polymer internalization, we envisioned using antigenic polymers equipped with a group that would report directly on internalization.Most approaches to following ligand internalization rely on appending a fluorescent dye to the molecule of interest. Uptake is detected using a discontinuous assay (typically fluorescence microscopy). Becaus...

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Multivalency

Cloninger¹,

Bilgiçer²,

Li³

et al. 2012

Supramolecular Chemistry

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Multivalent interactions, in which multiple ligands on one entity bind simultaneously to multiple receptors on another, are ubiquitous in nature. The use of multivalent agents will undoubtedly transform existing methods that target important disease processes including infection, immune system regulation, cancer, and neurological pathways. The employment of multivalent systems as tools to study biological recognition processes and for therapeutic development is unique in that multivalency can address many problems that traditional monovalent materials cannot. Here, mechanisms of multivalent ligand binding as well as the thermodynamic principals of multivalency including the chelation effect, cooperativity, avidity enhancement, avidity entropy, and effective molarity are described. Experimental techniques that are currently used to study multivalent interactions are briefly compared. Key examples of systems that have been used to study multivalent interactions are highlighted and range from small molecules and carbohydrate‐functionalized macromolecules to antibodies and polymers. Overall, this chapter focuses on strategic approaches and enabling technologies that are advancing the development of multivalent therapeutic agents.

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Activating B Cell Signaling with Defined Multivalent Ligands

Cited by 163 publications

References 76 publications

Sialylated multivalent antigens engage CD22in transand inhibit B cell activation

Sialylated multivalent antigens engage CD22in transand inhibit B cell activation

Synthesis of Fluorogenic Polymers for Visualizing Cellular Internalization

Multivalency

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