Loss of B3GAT1/HNK-1 disrupts glioma-CD8⁺ T cell immune synapse formation for immune escape

Glioma is an immunologically evasive tumor with a lymphocyte-deficient tumor immune landscape, suggesting an unknown failure in tumor-stroma interaction. The human natural killer-1 glycan (HNK-1) is present on neurons and immune cells, suggesting a potential source of glioma-stroma interaction, cross-talk, and immune regulation. Immunohistochemical staining of a human glioma microarray showed that HNK-1 progressively decreased with increasing tumor grade. However, similar immunohistochemical staining for β-1,3-glucuronic acid transferase (B3GAT1), the predominant enzyme responsible for HNK-1 synthesis, showed no change with glioma progression, indicating that the loss of HNK-1 was not due to changes in B3GAT1 expression. However, Kaplan-Meier analysis showed that B3GAT1 levels positively correlated with survival. In the syngeneic GL261 murine glioblastoma model, HNK-1 knockdown by two B3gat1 shRNAs accelerated glioma growth and reduced mouse survival in vivo. B3gat1-knockdown tumors had increased numbers of regulatory T cells, and decreased numbers of effector CD8⁺ T cells, which correlated with increased CD8⁺ T-cell apoptosis. In co-cultures of CD8⁺ T cells with B3gat1-knockdown GL261 cells, we observed reduced T-cell-induced glioma Ca²⁺ signaling and intracellular perforin accumulation, along with increased perforin release into the culture medium, compared to CD8⁺ T-cell co-culture with wild-type GL261 cells. FACS analysis showed loss of co-stimulatory CD80, an immune synapse component, following B3gat1 knockdown. These results suggest that loss of HNK-1 expression contributes to tumor immune escape through loss of immune recognition and attack via downregulation of tumor cell surface co-stimulatory molecules, leading to reduced CD8⁺ T-cell activation and immune synapse formation, and increased T-cell apoptosis.