Proximity-Gated Hybridization Chain Reaction for Minimally Perturbative In Situ Visualization of PD-L1 Glycosylation on Living Immune Cells

Abstract Glycosylation of programmed death ligand 1 (PD-L1) is an important regulator of the PD-1/PD-L1 immune checkpoint and may contribute to heterogeneous responses in cancer immunotherapy. However, analytical methods that enable protein-specific, amplified, and minimally perturbative interrogation of PD-L1 glycosylation in functional immune cells remain limited. Herein, we developed a function-preserving, proximity-gated hybridization chain reaction (HCR) strategy for in situ visualization of PD-L1 glycosylation on living cells. This strategy integrates metabolic glycan labeling (MGL) with aptamer-mediated PD-L1 recognition, enabling HCR amplification only when the glycan-labeling probe and the PD-L1-recognition probe are brought into close proximity at PD-L1-associated glycans. On living DC2.4 dendritic cells, the method achieved a 13.2-fold signal enhancement over the nonamplified proximity-imaging format and enabled sensitive tracking of enzymatically induced glycosylation changes. Notably, the proximity-gated HCR assembly preserved dendritic cell phenotype and PD-L1/PD-1 recognition capacity. Cross-cell-type applicability was further validated in macrophages and tumor cells. PD-L1 glycosylation on dendritic cells showed a strong positive association with PD-1 recognition. Extension to primary bone marrow-derived dendritic cells further linked PD-L1-associated glycosylation to suppressed CD8+ T-cell proliferation. Overall, this work establishes a sensitive, function-preserving analytical method for protein-specific glycosylation detection on living immune cells and provides a useful tool for studying glycosylation-dependent immunoregulation.