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

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.