Deciphering Cholesterol's Role in PD-L2 Stability: A Distinct Regulatory Mechanism From PD-L1

[Display omitted] •Elucidation of cholesterol’s integral role in stabilizing PD-L2.•NMR characterization of PD-L2-TC within lipid membranes.•Identification of a unique cholesterol-binding motif in PD-L2.•Offering fresh perspectives on the immune regulatory mechanism of PD-L2. Programmed cell death 1 ligand 2 (PD-L2), a member of the B7 immune checkpoint protein family, emerges as a crucial player in immune modulation. Despite its functional overlap with programmed cell death 1 ligand 1 (PD-L1) in binding to the programmed cell death protein 1 (PD-1) on T cells, PD-L2 exhibits a divergent expression pattern and a higher affinity for PD-1. However, the regulatory mechanisms of PD-L2 remain under-explored. Here, our investigations illustrate the pivotal role of cholesterol in modulating PD-L2 stability. Using advanced nuclear magnetic resonance (NMR) and biochemical analyses, we demonstrate a direct and specific binding between cholesterol and PD-L2, mediated by an F-xxx-V-xx-LR motif in its transmembrane domain, distinct from that in PD-L1. This interaction stabilizes PD-L2 and prevents its downstream degradation. Disruption of this binding motif compromises PD-L2′s cellular stability, underscoring its potential significance in cancer biology. These findings not only deepen our understanding of PD-L2 regulation in the context of tumors, but also open avenues for potential therapeutic interventions.