No abstract

The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created “sialic acid low” tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing “sialic acid low” tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack.

Cross-presentation is an important mechanism by which DCs present exogenous antigens on MHC-I molecules, and activate CD8 1 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-Lewis A 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-Lewis A 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 À/À and MyD88-TRIFF À/À bone marrow-derived DCs (BMDCs), we show that the crosspresentation of the neo-glycoconjugates is dependent on MR and independent of TLR-mediated signaling. Whereas proliferation of antigen-specific CD4 1 T cells was unchanged, stimulation with neo-glycoconjugate-loaded DCs enhanced the generation of IFN-c-producing T cells. We conclude that modification of antigen with either 3-sulfo-Lewis A or tri-GlcNAc enhances crosspresentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC-based vaccination strategies to treat cancer.

We recently showed that MGL2 specifically binds tumour-associated glycan N-acetylgalactosamine (GalNAc). We here demonstrate that modification of an antigen with tumour-associated glycan GalNAc, targets antigen specifically to the MGL2 on bone marrow derived (BM)-DCs and splenic DCs. Glycan-modification of antigen with GalNAc that mimics tumourassociated glycosylation, promoted antigen internalisation in DCs and presentation to CD4 T cells, as well as differentiation of IFN-c producing CD4 T cells. Furthermore, GalNAc modified antigen enhanced cross-presentation of both BM-DCs and primary splenic DCs resulting in enhanced antigen specific CD8 T cell responses. Using MyD88-TRIFF 2/2 BM-DCs we demonstrate that the enhanced cross-presentation of the GalNAc modified antigen is TLR independent. Our data strongly suggest that tumourassociated GalNAc modification of antigen targets MGL on DCs and greatly enhances both MHC class II and class I presentation in a TLR independent manner.Dendritic cells (DCs) are specialised antigen presenting cells (APC) well known for their capacity to induce MHC class I and MHC class II antigen presentation by stimulating antigen specific CD8 and CD4 T cells, respectively. Distinct classes of receptors are involved in processes that regulate recognition and uptake of antigen while others receptors induce DC maturation upon antigen recognition. The most commonly studied pattern (pathogen) recognition receptors (PRR) on DCs are the Toll like receptors (TLR) and C-type lectin receptors (CLR). TLR recognise microbial and endogenously conserved molecular patterns resulting in induction of intra-cellular signalling cascade. 1 Activation of TLR signalling cascade results in DC maturation and production of pro-inflammatory cytokines. On the other hand, CLR are endocytic receptors that recognise and internalise glycosylated pathogenic and self-antigens. Antigen internalisation facilitates the intra-cellular processing of antigen as well as loading on to MHC class II molecules and presentation to CD4 T cells leading for efficient T cell proliferation.2,3 Alternatively, CLR such as DEC-205, mannose receptor and CLEC9A have been shown to mediate antigen uptake leading to MHC class I loading and CD8 T cell priming, a process known as cross-presentation. 4-8 Crosspresentation is an important mechanism crucial for the induction of immune responses to viral infections and tumours. DCs are very efficient in cross-presenting antigens. In mice, splenic CD8a þ DCs are known to efficiently cross-present antigens to CD8 T cells. 10 The traditional view of cross-presentation states that antigen endocytosed by APC can be processed by at least 2 different mechanisms depending upon the nature of the antigen. Internalised antigen is exported from the phagosome into the cytoplasm for proteosomal processing, followed by TAP-dependent loading of antigen to MHC class I molecules in the endoplasmic reticulum or phagosomes. 11-13Alternatively, endocytosed antigen is cleaved in endocytic vesicles by proteases such as cat...

C 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 C 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 CD8C 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.