Tn-Glycoconjugates engage MGL2 to potentiate TLR9-mediated dendritic cell maturation and Th1-skewed immune responses

Abstract

Dendritic cells (DCs) orchestrate antitumor immunity by integrating signals from the tumor microenvironment to prime effective T cell responses. Many tumors display altered glycosylation patterns, including tumor-associated carbohydrate antigens (TACAs) such as the Tn antigen (GalNAcα1-O-Ser/Thr); yet how these structures influence DC function is not well defined. Here, we investigated how Tn-bearing glycoconjugates modulate DC activation and shape adaptive immunity. Bone marrow-derived DCs (BMDCs) efficiently internalized fluorescently labeled Tn-glycoconjugates, whereas uptake of non-glycosylated counterparts was negligible. Although Tn-glycoconjugates alone did not induce DC maturation, co-stimulation with the Toll-like receptor 9 (TLR9) agonist CpG markedly increased CD86 expression and the secretion of IL-12/23p40 and IL-6, with the multivalent construct MAG:Tn3-PV eliciting the strongest response. These conditioned BMDCs promoted strong IFN-γ production by allogeneic splenocytes, consistent with a Th1-polarizing phenotype. Mechanistically, both uptake and CpG-enhanced activation required the C-type lectin receptor MGL2, as blockade of MGL or competition with GalNAc abrogated glycoconjugate uptake and CpG-enhanced cytokine induction. Pharmacological inhibition revealed that MGL2 signaling synergizes with TLR9 through the Syk–Raf-1–NF-κB axis. In vivo, mice immunized with DCs conditioned with Tn-glycoconjugate- plus CpG displayed enhanced splenocyte proliferation, increased IFN-γ secretion, and elevated cytotoxic activity without IL-10 induction, confirming a Th1-skewed response. Collectively, these findings identify MGL2 as a critical mediator of Tn-glycoconjugate sensing and unveil a synergistic C-type lectin receptor (CLR)–TLR9 cross-talk that amplifies DC maturation and cytotoxic immunity. This study provides mechanistic insight into how specific glycan–lectin interactions fine-tune innate receptor signaling, highlighting the potential of Tn-based glycoconjugates as immunomodulatory tools for vaccine design and cancer immunotherapy.

Introduction

Dendritic cells (DCs) are pivotal orchestrators of immune responses, serving as professional antigen-presenting cells that bridge innate and adaptive immunity. Their capacity to capture, process, and present antigens to T cells is essential for immune surveillance and for the initiation of effective responses against pathogens and tumors. ¹ In cancer, the induction of robust antitumor immunity depends on the activation and maturation of DCs to elicit potent cytotoxic T lymphocyte (CTL) responses capable of eliminating malignant cells.^2,3

DC-based immunotherapy represents a promising strategy for cancer treatment, employing either ex vivo antigen loading or in vivo DC targeting. ⁴ Ex vivo approaches offer precise control over antigen selection, DC maturation, and the inclusion of immunostimulatory adjuvants that enhance cross-presentation and CD8⁺ T-cell activation, resulting in potent cytotoxic responses. ⁵ However, these protocols are labor-intensive, costly, and subject to interindividual variability.⁶ In contrast, in vivo DC targeting provides a simpler, faster, and less expensive alternative, allowing direct delivery of antigens to DCs. ⁷ Nonetheless, this approach offers less control over DC activation and maturation, potentially leading to suboptimal or off-target immune responses.^5,7

Tumor-associated carbohydrate antigens (TACAs) are aberrant glycan structures frequently expressed on the surface of malignant cells due to incomplete or altered glycosylation. These antigens, absent or minimally expressed in healthy tissues, are commonly exposed on mucins and other glycoproteins, making them attractive targets for cancer diagnosis and immunotherapy. ⁸ Among TACAs, the Tn antigen (GalNAcα1-O-Ser/Thr) is one of the most extensively studied. It represents a truncated O-glycan generated by premature termination of mucin-type glycosylation, often resulting from defects in C1GALT1 or its molecular chaperone COSMC.^9–11 The Tn antigen is overexpressed in various epithelial cancers, including breast, colon, and pancreatic carcinomas, and correlates with tumor progression, metastasis, and poor prognosis.^10,11 Due to its tumor-restricted expression and immunogenicity, Tn represents a promising biomarker and target for antibody- or vaccine-based immunotherapies.

Ex vivo loading of DCs with glycoconjugates carrying TACAs, such as the Tn antigen, has been investigated to potentiate antitumor immune responses. These glycans are recognized by lectin receptors on DCs, facilitating targeted uptake and enhancing T-cell activation. ¹² Indeed, DCs pulsed with Tn-glycosylated peptides have been shown to elicit stronger cytotoxic T-cell responses than the non-glycosylated counterparts in experimental tumor models. ¹³ Similarly, conjugation of tumor-associated antigens such as MUC1 with mannose, a ligand for C-type lectin receptors (CLRs), improves antigen uptake, presentation, and subsequent T-cell activation. ¹⁴ Moreover, glycosylated tumor peptides have been reported to enhance the capacity of DCs to prime CTLs, resulting in improved antitumor immunity. ¹⁵

The Multiple Antigenic Glycopeptide (MAG-Tn3) is a dendrimeric multivalent vaccine that combines the Tn antigen with a CD4⁺ T-helper epitope.^16,17 In mice and primates, MAG-Tn3 induces strong humoral and cellular immune responses, promoting the production of anti-Tn antibodies capable of recognizing and protecting against Tn-expressing tumor cells.^17–19 Mechanistically, MAG-Tn3 targets dermal DCs via the macrophage galactose-type lectin (MGL) receptor, driving follicular CD4⁺ T-cell activation, germinal center B-cell formation, and Th2 cytokine secretion, ultimately generating high titers of anti-Tn antibodies that bind to human tumor cells. ¹⁸ Furthermore, clinical trials have demonstrated that vaccination with MAG-Tn3 is well tolerated and induces robust, specific anti-Tn antibody responses in humans. ²⁰ However, the induction of cytotoxic cellular responses has not yet been fully evaluated. In the present study, we assessed the ability of Tn-carrying glycoconjugates to enhance Toll-like receptor (TLR)-mediated DC maturation and to induce cytotoxic immune responses in vivo.

Methodology

Tn-glycoconjugates

The MAG:Tn3-PV and its non-glycosylated counterpart MAP-PV were synthetized by solid-phase synthesis as previously described ¹⁶ and provided by Dr Sylvie Bay (Institut Pasteur, Paris, France). MAG:Tn3-PV bears several a-D-GalNAc residues displayed as trimeric clusters on a Ser-Thr-Thr motif. The sequence of the PV peptide is KLFAVWKITYKDT. ¹⁹ The MUC6-Tn glycoconjugate was obtained as previously described. ²¹ Briefly, the MUC6 recombinant protein (86 amino acids) was subjected to enzymatic glycosylation using UDP-Nacetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T). MUC6 (40–80 μM) was incubated with UDP-GalNAc (2 equivalents per Thr/Ser residue) and ppGalNAc-T1 (kindly given by Dr F. Piller), -T2, or -T7 in 50 mM 2-(N-morpholino)ethanesulfonic acid buffer pH 6.5, containing 15 mM MnCl₂ for 24 h at 37°C. The resulting MUC6-Tn glycoconjugate was purified, lyophilized, and characterized by mass spectrometry. ²¹

Glycoconjugates were subsequently labeled with Atto-647 (Merck/Sigma-Aldrich) according to the manufacturer's instructions. Briefly, amine-reactive Atto-647 NHS ester was dissolved in anhydrous DMSO and added to the glycoconjugates in bicarbonate buffer (pH 8.3) to enable covalent coupling to primary amines. After incubation in the dark at room temperature, excess dye was removed by dyalisis.

Antigen internalization by bone marrow-derived DCs (BMDC)

BMDCs were generated from bone marrow precursors from BALB/c mice. Mouse handling and experiments were carried out in compliance with institutional guidelines and regulations from the National Committee on Animal Research (CNEA, Uruguay) and approved by the Universidad de la República's Committee on Animal Research (CHEA Protocol number 070153-000543-14). Briefly, bone marrow cells from femurs and tibias were harvested and plated at a density of 2 × 10⁵ cells/ml in RPMI-1640 with 400 g/ml of glutamine (Capricorn, Ebsdorfergrund, Germany) complete medium containing 10% heat-inactivated fetal bovine serum (FBS, Capricorn Scientific, Ebsdorfergrund, Germany), 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin (Merk, Sigma-Aldrich, St Louis, MO, USA) supplemented with 1% of a GM-CSF-containing supernatant and cultured for 6–7 days at 37°C and 5% CO₂. After 3 days of culture, the medium was replaced. Cells were recovered on days 6 or 7, by flushing the plates with 5 mM EDTA in Phosphate-Buffered Saline (PBS). In vitro internalization of (glyco)peptides was analyzed by flow cytometry on BMDCs incubated (2.5 × 10⁵/well) with Alexa 647-labeled antigen for 1 h at 37°C in complete medium. For inhibition assays, cells were incubated with glycoconjugates in complete medium supplemented with 10 mM EDTA, that chelates Ca²⁺ needed for CLR-binding, ²² 10 μg/ml of anti-MGL mAb (ERMP23, Cedarlane Laboratoires Ltd) or GalNAc (1 mM) for 1 h. Analyses were perfomed in a Cyan Flow cytometer. A total of 10,000 CD11c⁺ events were collected and analyzed per sample. Prior to analysis, cell debris was excluded based on forward and side scatter parameters, and singlets were selected to avoid doublets. Cell viability by was assessed using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) across all culture conditions, confirming that none of the tested (glyco)peptides exerted cytotoxic effects on the cells.

DC maturation assays

BMDCs (5 × 10⁴/well) were cultured with serial dilutions of (glyco)peptides (1 μM) in the presence or absence of CpG (1 μg) and incubated overnight at 37°C. Alternatively, BMDCs were incubated with signaling inhibitors targeting Syk (BAY 61-3606, 5 μM), Raf-1 (GW5074, 10 μM), and NF-κB (Bay 11-7082, 5 μM). DC were stained with anti-CD11c-PE, CD40-FITC, and CD86-APC and the expression of costimulatory molecules was evaluated by flow cytometry. IL-6, IL-12/23p40, IL-10 and TNFα were quantified on the culture supernatants by ELISA.

The ability of BMDCs to activate allogenic lymphocytes was assessed using BMDCs generated from BALB/c female mice, and splenocytes isolated from female C57BL/6 mice. BMDCs were pre-stimulated, centrifuged, and the resulting supernatants were collected. Splenocytes were subjected to red blood cell lysis using lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 1 mM EDTA pH 7.2–7.4) for 10 min, then washed in PBS, and resuspended in complete medium. Cells were counted and co-cultured with BMDCs at a 5:1 ratio (splenocytes:BMDCs). Cultures were incubated at 37°C for 3 days, after which supernatants were collected and analyzed by ELISA to determine IFN-γ and IL-10 levels.

In vivo stimulatory capacity of conditioned BMDCs

BALB/c mice were inoculated intraperitoneally (i.p.) with 1 × 10⁶ BMDCs previously conditioned with MAG-Tn-3PV or MAP-PV in the presence of CpG as previously described. The procedure was repeated 10 days later. For conditioning, BMDCs were incubated with medium alone, MAG:Tn3-PV, or MAP-PV at 0.1 μM, and all groups received CpG at 0.3 μg/ml for 18 h at 37°C. Ten days after the second inoculation, animals were sacrificed and spleens were harvested for splenocyte isolation. Splenocytes were re-stimulated with ConA (5 μg/ml), or PV peptide at 2 μM for 3 days at 37°C. Culture supernatants were subsequently analyzed to measure IL-10 and IFN-γ levels.

In vitro cytotoxic capacity of splenocytes from mice inoculated with glycoconjugate-conditioned BMDCs

Splenocytes from mice inoculated with conditioned BMDCs (as described above) were collected and co-cultured overnight with Tn-expressing TA3/Ha tumor cells. Various effector-to-target (E:T) ratios were tested: 25:1 and 50:1. As a 100% cytotoxicity control, TA3/Ha cells were incubated with 1% Triton X-100. Controls of splenocytes alone and tumor cells alone were also included. The next day, 10 μL of WST-8 reagent (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt, Sigma-Aldrich, St Louis, MO) was added to each well and incubated at 37°C for 5 h. Absorbance was then measured at 460 nm and 650 nm, and the percentage of cytotoxicity was calculated using the following formula:

$$\text{\%}Cytotoxicity={\displaystyle \frac{Salone+Talone-(E+T)}{Talone-TTritonX100}\times 100}$$

Where: S alone: Absorbance ratio 460 nm / 650 nm of wells with splenocytes only; T alone: Absorbance ratio 460 nm / 650 nm of wells with tumor cells only: S + T: Absorbance ratio 460 nm / 650 nm of wells with the mixture of tumor cells and splenocytes; T TritonX100: Absorbance ratio 460 nm / 650 nm of wells with tumor cells incubated with 1% Triton X-100.

Results and discussion

Because internalization is a key first step in DC sensing and antigens, we first evaluated the capacity of DCs to internalize the glycoconjugates MAG:Tn-PV and MUC6:Tn. BMDCs were incubated with fluorescently labeled Tn-glycoconjugates or non-glycosylated counterparts at 37°C to assess internalization. As shown in Figure 1A, BMDCs efficiently internalized Tn-glycoconjugates, whereas uptake of non-glycosylated counterparts MAP-PV and MUC6 was negligible under the tested conditions. We then examined whether Tn-glycoconjugates could induce DC maturation or modulate CpG-induced activation. Tn-glycoconjugates alone did not induce maturation, as expression of costimulatory molecules and cytokines remained unaltered (Figure 1B–C). In contrast, when combined with CpG, Tn-glycoconjugates enhanced DC activation, evidenced by increased CD86 expression (Figure 1B) and elevated production of IL-12/23p40 and IL-6 (particularly for MAG:PV-Tn3-PV) (Figure 1C). Notably, the non-glycosylated peptide MAP:PV inhibited expression of CD40, CD86, and IL-6, whereas TNFα and IL-12/23p40 remained unchanged and IL-10 was undetectable. This opposite pattern relative to the Tn-modified counterparts indicates that glycosylation is not merely additive but actively redirects DC signaling toward an activating rather than inhibitory profile. Functionally, BMDCs conditioned with Tn-glycoconjugates plus CpG displayed enhanced stimulatory capacity toward allogeneic splenocytes, resulting in elevated IFN-γ secretion than CpG stimulation alone, while IL-10 levels remained minimal (Figure 1D).

Figure 1.

Tn-Glycosylation facilitates antigen uptake and potentiates CpG-driven maturation and allostimulatory function. (A) BMDCs were incubated with fluorescently labeled Tn-glycosylated (MAG:Tn-PV, MUC6:Tn) or non-glycosylated counterparts (MAP-PV, MUC6) for 1 h at 37°C, and internalization was analyzed by flow cytometry in CD11c⁺ cells. (B-C) BMDCs were stimulated with Tn-glycoconjugates (1 μM) in the presence or absence of CpG (0.1 μg) for 24 h, and expression of costimulatory molecules (CD40, CD86) and cytokine production (IL-12/23p40, IL-6, TNF-α, IL-10) were measured by flow cytometry and ELISA, respectively. (D) Conditioned BMDC were washed and cultured with allogeneic splenocytes (ratio splenocytes:BMDC: 5:1) for 3 days and IFN-γ and IL-10 secretion was quantified by ELISA. Data are representative of at least two independent experiments. Asterisks represent significant differences calculated with one way Anova with pく0.05.

These results demonstrate that Tn-glycoconjugates are efficiently internalized by DCs and can potentiate CpG-driven maturation. This is consistent with previous observations that TACAs, including the Tn antigen, are recognized by CLRs on DCs, facilitating antigen uptake and presentation. ¹⁸ Carbohydrate-lectin interactions are established endocytic routes into DCs, and Tn-bearing structures are known ligands of MGL, a CLR specific for Gal/GalNAc residues.^23–26 The increase in IL-12/23p40 and IL-6, together with CD86 upregulation and the absence of IL-10, suggests a polarization toward a proinflammatory, Th1-promoting phenotype.^27,28 Similar effects have been reported with carbohydrate-modified antigens that enhance DC immunogenicity and promote IFN-γ production by T cells.^24,29,30 The differences observed between the two Tn-glycoconjugates indicate that the peptide context (Serine versus Threonine, proximal amino acids), the Tn density, the steric hindrance or flexibility of the carbohydrate environment (N-terminal display for MAG versus intra-chain for MUC6) or antigen display may modulate lectin engagement and downstream signaling. Overall, these findings support a role for Tn glycosylation in fine-tuning innate receptor signaling and thereby modulating DC-mediated immune activation.

To identify the receptor involved in the described DC activation by Tn, we next assessed the contribution of MGL2 (Macrophage Galactose-type Lectin 2), which recognizes exposed GalNAc/Gal moieties, including those of the Tn antigen, and can exert either tolerogenic or immunostimulatory effects depending on context. As shown in Figure 2A, internalization of Tn-glycoconjugates by BMDCs was inhibited by MGL blockade, EDTA, and GalNAc competition, demonstrating dependence on MGL-carbohydrate interaction. MGL engagement was also required for CpG-induced maturation: when MGL was blocked, CpG-induced IL-12/23p40 production was decreased (Figure 2B) and reduced the ability of BMDCs to stimulate IFN-γ secretion by splenocytes (Figure 2C). Notably, the anti-MGL antibody fully abolished the effect of MAG:Tn3-PV (Figure 2B). To determine whether specific signaling pathways contribute to the response elicited by MAG:Tn₃-PV, we used pharmacological inhibitors targeting Raf-1, Syk, and NF-κB. Inhibition of these pathways reduced MAG:Tn₃-PV–induced IL-12/23p40 and IL-6 production in CpG-stimulated BMDCs (Figure 2D), as well as IFN-γ secretion by allogeneic splenocytes (Figure 2E), indicating that these pathways are required for the full activation response. Surprisingly, non-glycosylated MAP-PV inhibited the CpG-induced production of IL-12/23p40 and IL-6 (Figure 2D) but not the IFN-γ produced in the MLR assay (Figure 2E), suggesting that MAP-PV targets signaling pathways that control DC inflammatory cytokine production while leaving intact the pathways required for promoting allogeneic T-cell responses.

Figure 2.

MGL mediates uptake and immunomodulatory signaling induced by Tn-glycopeptides in DCs. (A) Uptake of fluorescently labeled Tn-glycoconjugates (MAG:Tn-PV, MUC6:Tn) by BMDCs was analyzed in the presence of blocking anti-MGL antibodies, EDTA, or free GalNAc. (B) IL-12/23p40 secretion by glycoconjugate/CpG-conditioned BMDC in the presence of blocking anti-MGL antibodies, EDTA, or free GalNAc. (C) IFN-γ secretion by allogeneic splenocytes stimulated with conditioned BMDC in the presence of blocking anti-MGL antibodies, EDTA, or free GalNAc. (D–E) Cytokine secretion by BMDCs (D) or splenocytes (E) under the treatment with pharmacological inhibitors targeting Syk, Raf-1, or NF-κB. None corresponds to the basal condition. Data are representative of at least two independent experiments. Asterisk represent significant differences calculated with one way Anova with pく0.05. nd: not determined.

These results identify MGL2 as a key mediator of Tn-glycoconjugate internalization and immunomodulatory signaling in DCs. In the presence of CpG, a TLR9 agonist, MGL2 signaling synergizes with TLR9 pathways, likely through the Syk–Raf-1–NF-κB axis, consistent with prior reports of CLR–TLR cross-talk amplifying cytokine production and costimulatory molecule expression.^26,31 Previous studies using human MUC1-Tn glycoconjugates also demonstrated extracellular signal-regulated kinase (ERK) and NF-κB activation downstream of MGL engagement. ³² Our findings therefore reinforce the concept that MGL-Tn interaction can enhance TLR9-driven maturation and shape DC function via shared intracellular signaling nodes.^33,34 Of note, the inhibitory effect of MAP-PV in the production of proinflamatory cytokines induced by CpG should be studied in depth, since no previous studies have investigated the effect of this CD4⁺-T cell epitope in TLR-signaling on BMDCs.

Finally, we investigated whether BMDCs conditioned with Tn-glycoconjugates plus CpG could drive Th1 differentiation and proliferation in vivo. BMDCs were incubated with Tn-glycoconjugates and CpG for 18 h and injected intraperitoneally into mice. One week later, splenocytes were restimulated with either the T-cell epitope PV or ConA. Tn-glycoconjugate conditioning induced enhanced IFN-γ production (Figure 3A), consistent with a Th1 response, whereas IL-10 levels did not increase (Figure 3B). This was accompanied by increased cytotoxic activity (Figure 3C), as measured by co-culturing splenic T cells from mice that received Tn-glycoconjugate–treated DCs with tumor target cells, indicating that Tn-glycoconjugate treatment augments CpG-driven adaptive activation.

Figure 3.

Tn-glycopeptide-conditioned DCs promote Th1 polarization and cytotoxic immune responses in vivo. (A–B) BMDCs were pulsed with Tn-glycoconjugates and CpG or controls (saline or CpG alone), injected intraperitoneally into mice, and splenocytes were collected one week later. Cells were restimulated with PV peptide, ConA or medium (Med) for 3 days at 37°C, and IFN-γ (A) and IL-10 (B) secretion was quantified by ELISA. (C) Splenocytes from mice immunized with Tn-glycopeptide/CpG-conditioned DCs were co-cultured with TA3/Ha Tn⁺ tumor cells at splenocyte-to-tumor cell ratios of 1:1 or 5:1. Tumor cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Asterisk represent significant differences calculated with one way Anova with p < 0.05.

Taken together, these data demonstrate that Tn-glycoconjugates, through MGL2 engagement, synergize with TLR9 signaling to promote DC maturation and Th1-skewed immune responses.^35–37 The resulting proinflammatory cytokine milieu and cytotoxic activity suggest that MGL-dependent sensing of glycosylated antigens can enhance type 1 immunity. However, it also should be taken into account that abnormal display of protein glycosylation can actively influence how the immune system recognizes and responds to tumor-associated molecules. Indeed, glycosylation can also shape the nature of the immune response. ³⁸ These findings identify tumor-associated glycosylation patterns as functional modulators of innate signaling, providing a conceptual and mechanistic basis for developing glycoconjugate vaccines or adjuvants that selectively enhance type 1 immunity.