Changes in glycosylation during tumour progression are a key hallmark of cancer. One of the glycan moieties generally overexpressed in cancer are sialic acids, which can induce immunomodulatory ...properties via binding to Siglec receptors. We here show that Pancreatic Ductal Adenocarcinoma (PDAC) tumour cells present an increased sialylation that can be recognized by Siglec-7 and Siglec-9 on myeloid cells. We identified the expression of the α2,3 sialyltransferases ST3GAL1 and ST3GAL4 as main contributor to the synthesis of ligands for Siglec-7 and Siglec-9 in tumour cells. Analysing the myeloid composition in PDAC, using single cell and bulk transcriptomics data, we identified monocyte-derived macrophages as contributors to the poor clinical outcome. Tumour-derived sialic acids dictate monocyte to macrophage differentiation via signalling through Siglec-7 and Siglec-9. Moreover, triggering of Siglec-9 in macrophages reduce inflammatory programmes, while increasing PD-L1 and IL-10 expression, illustrating that sialic acids modulate different myeloid cells. This work highlights a critical role for sialylated glycans in controlling immune suppression and provides new potential targets for cancer immunotherapy in PDAC.
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•There is multilevel complexity in glycan-induced immune modulation.•Lectin binding is dependent on glycan constitution and carrier properties.•Multivalent presentation of glycans is ...key to novel biotechnological designs.•More insight in multivalent glycan-induced lectin signalling is necessary.
Glycans play a crucial role to discern between self and foreign entities by providing key recognition elements for C-type lectin receptors (CLRs) and Siglec receptors expressed on immune cells. The glycan recognition of CLRs has illustrated a potent immune modulatory role affecting not only innate pathogen binding and immune signalling, but also Thelper differentiation, cytokine production and antigen presentation. This broad range of influence has implicated glycans in the pathogenesis of infectious diseases but also revealed their extraordinary properties in cancer. Glycan binding by CLRs and Siglecs can be exploited for immunotherapy and the design of glycan-based therapeutics and their multivalent requirements will aspire new biotechnological approaches to effectively interfere in immunological processes in cancer and infectious diseases.
Many tumors display alterations in the biosynthetic pathways of glycosylation, resulting in increased expression of specific tumor-associated glycan structures. Expression of these altered glycan ...structures is associated with metastasis and poor prognosis. Antigen presenting cells can recognize tumor-associated glycan structures, including the truncated
-glycan Tn antigen, via specific glycan receptors. Tn antigen-mediated activation of the C-type lectin MGL on dendritic cells induces regulatory T cells via the enhanced secretion of IL-10. Although these findings indicate that MGL engagement by glycan ligands can modulate immune responses, the impact of MGL ligation on dendritic cells is still not completely understood. Therefore, we employed RNA sequencing, GO term enrichment and pathway analysis on human monocyte-derived dendritic cells stimulated with two different MGL glycan ligands. Our analyses revealed a reduced expression of genes coding for key enzymes involved in the glycolysis pathway, TCA cycle, and oxidative phosphorylation. In concordance with this, extracellular flux analysis confirmed the decrease in glycolytic activity upon MGL triggering in human dendritic cells. To our knowledge, we are the first to report a diminished glycolytic activity of human dendritic cells upon C-type lectin stimulation. Overall, our findings highlight the impact of tumor-associated glycans on dendritic cell biology and metabolism and will increase our understanding on how glycans can shape immunity.
Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through ...modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8+ T cells in vitro and in vivo. CD8+ T cells from mice vaccinated with trimers showed an KLRG1+ effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform , termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8+ and CD4+ T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8+ T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8+ T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity.
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Dendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation of ...DCs is key, as well as the production of cytokines that help to accomplish T cell responses. DC-SIGN is a frequently exploited PRR that can effectively be targeted with mannosylated antigens to enhance the induction of antigen-specific T cells. The natural
-mannosidic linkage is susceptible to enzymatic degradation, and its chemical sensitivity complicates the synthesis of mannosylated antigens. For this reason, (oligo)mannosides are generally introduced in a late stage of the antigen synthesis, requiring orthogonal conjugation handles for their attachment. To increase the stability of the mannosides and streamline the synthesis of mannosylated peptide antigens, we here describe the development of an acid-stable
-mannosyl lysine, which allows for the inline introduction of mannosides during solid-phase peptide synthesis (SPPS). The developed amino acid has been successfully used for the assembly of both small ligands and peptide antigen conjugates comprising an epitope of the gp100 melanoma-associated antigen and a TLR7 agonist for DC activation. The ligands showed similar internalization capacities and binding affinities as the
-mannosyl analogs. Moreover, the antigen conjugates were capable of inducing maturation, stimulating the secretion of pro-inflammatory cytokines, and providing enhanced gp100 presentation to CD8
and CD4
T cells, similar to their
-mannosyl counterparts. Our results demonstrate that the
-mannose lysine is a valuable building block for the generation of anticancer peptide-conjugate vaccine modalities.
House dust mite extract–based allergen immunotherapy (AIT) to treat house dust mite allergy is substantially effective but still presents some safety and efficacy concerns that warrant improvement. ...Several major allergen-based approaches to increase safety and efficacy of AIT have been proposed. One of them is the use of the group 2 allergen, Der p 2.
We sought to investigate the immunomodulatory effects of sialic acid–modified major allergen recombinant Der p 2 (sia-rDer p 2) on PBMCs from healthy volunteers.
We activated PBMCs with anti-CD3/CD28 antibodies and incubated them at 37°C for 6 days in the presence or absence of either native rDer p 2 or α2-3 sialic acid–modified rDer p 2 (sia-rDer p 2). We assessed the changes in CD4+ T-cell activation and proliferation by flow cytometry and changes in T-lymphocyte cytokine production in cell culture supernatant by ELISA.
We observed that PBMCs treated with sia-rDer p 2 presented with a markedly decreased expression of CD69 and an increased abundance of LAG-3+ lymphocytes compared with cells treated with rDer p 2. Moreover, PBMCs treated with sia-rDer p 2 showed a reduced production of IL-4, IL-13, and IL-5 and displayed a higher IL-10/IL-5 ratio compared with rDer p 2–treated PBMCs.
We demonstrate that sia-rDer p 2 might be a safer option than native rDer p 2 for Der p 2–specific AIT. This is most relevant in the early phase of AIT that is often characterized by heightened TH2 responses, because sia-rDer p 2 does not enhance the production of TH2 cytokines.
Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune ...responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.
Dendritic cell (DC)–targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and ...presentation to CD4+ and CD8+ T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node–resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use.
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour ...progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.
Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone ...marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U
A and C
A sequences (endoY
A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoU
) or 2'-5' phosphodiesterase (PDE
) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U
A and C
A sequences (endoY
A) within viral (+) strand RNA to evade dsRNA-activated host responses.
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