Evading host immune surveillance is one of the hallmarks of cancer. Immune checkpoint therapy, which aims to eliminate cancer progression by reprogramming the antitumor immune response, currently occupies a solid position in the rapidly expanding arsenal of cancer therapy. As most immune checkpoints are membrane glycoproteins, mounting attention is drawn to asking how protein glycosylation affects immune function. The answers to this fundamental question will stimulate the rational development of future cancer diagnostics and therapeutic strategies.

Leveraging the T cell immunity against tumors represents a revolutionary type of cancer therapy. 4-1BB is a well-characterized costimulatory immune receptor existing on activated T cells and mediating their proliferation and cytotoxicity under infectious diseases and cancers. Despite the accumulating interest in implementing 4-1BB as a therapeutic target for immune-related disorders, less is known about the pattern of its intracellular behaviors and regulations. It has been previously demonstrated that 4-1BB is heavily modified by N-glycosylation; however, the biological importance of this modification lacks detailed elucidation. Through biochemical, biophysical, and cell-biological approaches, we systematically evaluated the impact of N-glycosylation on the ligand interaction, stability, and localization of 4-1BB. We hereby highlighted that N-glycan functions by preventing the oligomerization of 4-1BB, thus permitting its membrane transportation and fast turn-over. Without N-glycosylation, 4-1BB could be aberrantly accumulated intracellularly and fail to be sufficiently inserted in the membrane. The N-glycosylation-guided intracellular processing of 4-1BB serves as the potential mechanism explicitly modulating the “on” and “off” of 4-1BB through the control of protein abundance. Our study will further solidify the understanding of the biological properties of 4-1BB and facilitate the clinical practice against this promising therapeutic target.