W.W.J. Unger

Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells

Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells in order to achieve an appropriate uptake, processing, and presentation to Ag-specific T cells. C-type lectins have shown to be ideal receptors for the targeting of antigens to dendritic cells and allow the use of their natural ligands - glycans - instead of antibodies. Amongst them, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is an interesting candidate due to its biological properties and the availability of its natural carbohydrate ligands. Using Le(b)-conjugated poly(amido amine) (PAMAM) dendrimers we aimed to characterize the optimal level of multivalency necessary to achieve the desired internalization, lysosomal delivery, Ag-specific T cell proliferation, and cytokine response. Increasing DC-SIGN ligand multivalency directly translated in an enhanced binding, which might also be interesting for blocking purposes. Internalization, routing to lysosomal compartments, antigen presentation and cytokine response could be optimally achieved with glycopeptide dendrimers carrying 16-32 glycan units. This report provides the basis for the design of efficient targeting of peptide antigens for the immunotherapy of cancer, autoimmunity and infectious diseases.

'Dressed for success' C-type lectin receptors for the delivery of glyco-vaccines to dendritic cells

Current strategies in immunotherapy for the treatment of tumors or autoimmunity focus on direct in vivo targeting of antigens to dendritic cells (DC), as these cells are the key regulators of immune responses. Multiple DC subsets can be distinguished in both humans and mice, based on phenotype and location. Moreover, recent data show that these subsets have distinct functions. All these features have implications for the design of DC-targeting vaccines. In this review we integrate recent knowledge on the different DC subsets in human and mice and how DC-expressed C-type lectin receptors (CLR) can be exploited for the induction of either antigen-specific immunity or tolerance.

CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG

Human myelin oligodendrocyte glycoprotein is decorated with fucosylated N-glycans that are recognized by DC-SIGN+ DCs and microglia that control immune homeostasis.

Design of neo-glycoconjugates that target the mannose receptor and enhance TLR-independent cross-presentation and Th1 polarization.

Cross‐presentation is an important mechanism by which DCs present exogenous antigens on MHC‐I molecules, and activate CD8+ T cells, cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross‐presentation of tumor antigens and Th polarization, processes that are pivotal for the anti‐tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3‐sulfo‐LewisA and tri‐GlcNAc (N‐acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3‐sulfo‐LewisA or tri‐GlcNAc specifically directs antigen to the MR. Both neo‐glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross‐presentation. Using MR−/− and MyD88‐TRIFF−/− bone marrow‐derived DCs (BMDCs), we show that the cross‐presentation of the neo‐glycoconjugates is dependent on MR and independent of TLR‐mediated signaling. Whereas proliferation of antigen‐specific CD4+ T cells was unchanged, stimulation with neo‐glycoconjugate‐loaded DCs enhanced the generation of IFN‐γ‐producing T cells. We conclude that modification of antigen with either 3‐sulfo‐LewisA or tri‐GlcNAc enhances cross‐presentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC‐based vaccination strategies to treat cancer.

Antigen targeting to dendritic cells combined with transient regulatory T cell inhibition results in long-term tumor regression

Therapeutic vaccinations against cancer are still largely ineffective. Major caveats are inefficient delivery of tumor antigens to dendritic cells (DCs) and excessive immune suppression by Foxp3+ regulatory T cells (Tregs), resulting in defective T cell priming and failure to induce tumor regression. To circumvent these problems we evaluated a novel combinatorial therapeutic strategy. We show that tumor antigen targeting to DC-SIGN in humanized hSIGN mice via glycans or specific antibodies induces superior T cell priming. Next, this targeted therapy was combined with transient Foxp3+ Treg depletion employing hSIGNxDEREG mice. While Treg depletion alone slightly delayed B16-OVA melanoma growth, only the combination therapy instigated long-term tumor regression in a substantial fraction of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8+ T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity.

Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001

Notch receptors and ligands mediate barrier function in brain endothelial cells

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by immune cell entry into the brain. In normal conditions, this infiltration of immune cells is prevented by the blood–brain barrier (BBB) which is characterized by highly specialized endothelial cells. Therefore, the BBB is of high importance for brain protection and homeostasis. However, during inflammatory processes, like the ones observed in the course of MS, the BBB is dysfunctional and leaky, allowing the entry of serum proteins and immune cells into the brain, aggravating lesion formation. Notch signaling is a pathway essential for proper vascular development; however its role during adult vascular homeostasis and during inflammation is not clearly understood. Several research groups have shown that during inflammatory conditions Notch signaling is altered in endothelial cells, hinting for a function of this pathway during inflammation. Therefore, we hypothesize that Notch signaling is involved in BBB function during homeostasis and during inflammatory processes. Here we show that inflammation alters Notch receptors and ligand expression in brain endothelial cells. Furthermore, when Notch signaling is inhibited, brain endothelial cells show a decrease in barrier function as demonstrated by decreased resistance and increased permeability of endothelial monolayers to fluorescent molecules. During inflammatory conditions we observe an even more pronounced effect, demonstrating the importance of Notch signaling in coping with inflammation. These results were also confirmed by the Notch receptors knockdown in brain endothelial cells. In addition, our results show that Notch ligands are important during migration processes of T cells through the brain vasculature. Taken together our results demonstrate the importance of Notch signaling in BBB homeostasis and function. We believe that by understanding how BBB function is regulated, we can develop targeted therapies to the brain vasculature and restore its function during disease.