Table 1_MGAT1 knockout in human dendritic cells enhance CD8+ T cell activation.docx

Dendritic cells (DCs) are crucial in regulating immune responses, making them a compelling target for immunotherapy. While DC vaccines have demonstrated safety and feasibility, their limited clinical efficacy underscores the need for strategies to enhance DC functionality. Emerging evidence highlights the regulatory roles of sialoglycans in DC biology, yet the structure-function relationships of other glycans remain poorly understood. To aid the understanding of DC glycobiology, we recently developed and validated a human model system based on genetically glycoengineered MUTZ-3-derived DCs and showed that ST6GAL1-mediated α2,6-sialylation specifically modulates CD4+ T cell activation. In this study, we knocked-out (KO) mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1) to investigate how the shift from complex to oligomannose N-glycans affects DC biology and function. MGAT1 KO completely abolished the synthesis of complex and hybrid N-glycans. Differentiation of MGAT1 KO MUTZ-3 cells into immature DCs (iDCs) induced upregulation of DC markers including CD1a, CD80, CD86, CCR6, and CD209, comparable to the upregulation observed in WT iDCs. Interestingly, MGAT1 KO iDCs displayed an enhanced immunostimulatory profile, marked by increased surface densities of CD40, HLA-ABC, and HLA-DR, in combination with elevated mRNA levels of NFKB1 and IFNB1. Consistent with this profile, MGAT1 KO iDCs highly enhanced the activation and proliferation of allogeneic human CD8+ T cells in vitro, resulting in significantly higher levels of secreted proinflammatory cytokines compared to WT iDCs. This enhanced CD8+ T cell activation persisted under PD-L1 blockade, underscoring the robustness of the MGAT1 KO–driven effect. Significantly elevated NFKB1 levels in the MGAT1 KO iDCs suggest enhanced NF-κB activity driving HLA and costimulatory molecule upregulation and robust CD8+ T cell activation. We further demonstrate that MGAT1 KO promotes accelerated DC differentiation, yielding DCs that after three days of differentiation acquire the capacity to activate T cells. Building on previous research into sialic acids in DC biology, our findings reveal a regulatory role for complex and hybrid N-glycans and specifically demonstrate how sialic acids on N-glycans influence distinct functional outcomes in T cell activation. Our findings support cell-based glycoengineering as an effective strategy to improve DC-based immunotherapies.