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Presentation Modality of Glycoconjugates Modulates Dendritic Cell Phenotype.

ABSTRACT: The comparative dendritic cell (DC) response to glycoconjugates presented in soluble, phagocytosable, or non-phagocytosable display modalities is poorly understood. This is particularly problematic, as the probing of immobilized glycans presented on the surface of microarrays is a common screen for potential candidates for glycan-based therapeutics. However, the assumption that carbohydrate-protein interactions on a flat surface can be translatable to development of efficacious therapies, such as vaccines, which are delivered in soluble or phagocytosable particles, has not been validated. Thus, a preliminary investigation was performed in which mannose or glucose was conjugated to cationized bovine serum albumin and presented to DCs in soluble, phagocytosable, or non-phagocytosable display modalities. The functional DC response to the glycoconjugates was assessed via a high throughput assay. Dendritic cell phenotypic outcomes were placed into a multivariate, general linear model (GLM) and shown to be statistically different amongst display modalities when comparing similar surface areas. The GLM showed that glycoconjugates that were adsorbed to wells were the most pro-inflammatory while soluble conjugates were the least. DC interactions with mannose conjugates were found to be calcium dependent and could be inhibited via anti-DC-SIGN antibodies. The results of this study aim to resolve conflicts in reports from multiple laboratories showing differential DC profiles in response to similar, if not identical, ligands delivered via different modalities. Additionally, this study begins to bridge the gap between microarray binding data and functional cell responses by highlighting the phenotypes induced from adsorbed glycoconjugates as compared to those in solution or displayed on microparticles.

SUBMITTER: Hotaling NA

PROVIDER: S-EPMC4486352 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Presentation Modality of Glycoconjugates Modulates Dendritic Cell Phenotype.

Hotaling N A NA Ratner D M DM Cummings R D RD Babensee J E JE

Biomaterials science 20141001 10

The comparative dendritic cell (DC) response to glycoconjugates presented in soluble, phagocytosable, or non-phagocytosable display modalities is poorly understood. This is particularly problematic, as the probing of immobilized glycans presented on the surface of microarrays is a common screen for potential candidates for glycan-based therapeutics. However, the assumption that carbohydrate-protein interactions on a flat surface can be translatable to development of efficacious therapies, such a ...[more]

PMID: 26146546

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Project description:Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with ?-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a ?1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated ?-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.

Dataset Information

Presentation Modality of Glycoconjugates Modulates Dendritic Cell Phenotype.

Publications

Presentation Modality of Glycoconjugates Modulates Dendritic Cell Phenotype.

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