Homodimerized cytoplasmic domain of PD-L1 regulates its complex glycosylation in living cells.

Whether membrane-anchored PD-L1 homodimerizes in living cells is controversial. The biological significance of the homodimer waits to be expeditiously explored. However, characterization of the membrane-anchored full-length PD-L1 homodimer is challenging, and unconventional approaches are needed. By using genetically incorporated crosslinkers, we showed that full length PD-L1 forms homodimers and tetramers in living cells. Importantly, the homodimerized intracellular domains of PD-L1 play critical roles in its complex glycosylation. Further analysis identified three key arginine residues in the intracellular domain of PD-L1 as the regulating unit. In the PD-L1/PD-L1-3RE homodimer, mutations result in a decrease in the membrane abundance and an increase in the Golgi of wild-type PD-L1. Notably, PD-1 binding to abnormally glycosylated PD-L1 on cancer cells was attenuated, and subsequent T-cell induced toxicity increased. Collectively, our study demonstrated that PD-L1 indeed forms homodimers in cells, and the homodimers play important roles in PD-L1 complex glycosylation and T-cell mediated toxicity.