Glycan-Lectin duet, Novel strategies for DC-targeting

Cross-presentation is an important mechanism by which dendritic cells (DC) present exogenous antigens in MHC-I molecules, and activate CD8+ T cells, which are crucial for the elimination of tumours. Our aim is to exploit the cross-presentation capacity of the Mannose Receptor (MR) to improve presentation of tumour-antigens, as well as T-helper cell polarisation, both of which are pivotal for anti-tumour potency of cytotoxic T cells. To this end, we selected two glycanligands of the MR, 3-sulfo-Lewis A and tri-GlcNAc, to conjugate the model antigen OVA and assessed the effect on antigen presentation and T-helper cell differentiation. Our results demonstrate that conjugation of either 3-sulfo-Lewis A or tri-GlcNAc specifically directs antigen to the MR. Both neo-glycoconjugates showed even at low doses, improved uptake compared to native OVA, resulting in enhanced cross-presentation. Using MR-/and MyD88-/BM-DC, we show that the cross-presentation of the neo-glycoconjugates is dependent on MR and independent of TLR-mediated signalling. Whereas proliferation of antigen-specific CD4+ T cells was unchanged, stimulation with neo-glycoconjugate-loaded DC improved generation of IFN-γ producing T cells. We conclude that modification of antigen with either 3-sulfo-Lewis A 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. Introduction Activation of antigen-specific cytotoxic T cells is crucial for the induction of adequate antitumour immunity. Since most tumour cells are poorly immunogenic, optimal presentation of tumour-derived antigens in MHC class I molecules on the surface of antigen presenting cells is required. An important mechanism that allows dendritic cells (DCs) to present exogenous antigens, such as tumour-derived antigens, in MHC class I molecules is cross-presentation1. At tumour lesions, multiple factors and cells are present that prevent the proper activation of DC that enter the lesion to sample for antigens2,3. Consequently, these DC will not be able to properly activate antigen-specific CD8+ T cells in the tumour-draining lymph nodes. To obtain therapeutic anti-tumour immunity powerful vaccination protocols are required. Current strategies focus either on ex-vivo loading of autologous DC as well as specifically targeting of antigens to DC in vivo. These new therapies may be combined with a regulatory T cell (Treg) depletion regimen, as these cells have been shown to block anti-tumour immune responses3-6. As a classical C-type lectin receptor (CLR), the Mannose Receptor (MR) binds carbohydrate structures such as mannose, fucose and N-acetylglucosamine (GlcNAc) in a calciumdependent manner7,8. Besides these carbohydrate structures, the MR has recently also been reported to bind sulfated sugars, such as sulfated oligosaccharides of the blood group antigens Lewis A (LeA) and Lewis X (LeX)8-10. Binding of these types of ligands occurs via the Cysteinerich (CR) region of the MR and in a calcium-independent manner8. The MR has been proposed to mediate antigen uptake and presentation by DC based on the finding that mannosylated proteins are presented more efficiently than non-mannosylated