Ligand-induced ubiquitination unleashes LAG3 immune checkpoint function by hindering membrane sequestration of signaling motifs.

Lymphocyte activation gene 3 (LAG3) has emerged as a promising cancer immunotherapy target, but the mechanism underlying LAG3 activation upon ligand engagement remains elusive. Here, LAG3 was found to undergo robust non-K48-linked polyubiquitination upon ligand engagement, which promotes LAG3's inhibitory function instead of causing degradation. This ubiquitination could be triggered by the engagement of major histocompatibility complex class II (MHC class II) and membrane-bound (but not soluble) fibrinogen-like protein 1 (FGL1). LAG3 ubiquitination, mediated redundantly by the E3 ligases c-Cbl and Cbl-b, disrupted the membrane binding of the juxtamembrane basic residue-rich sequence, thereby stabilizing the LAG3 cytoplasmic tail in a membrane-dissociated conformation enabling signaling. Furthermore, LAG3 ubiquitination is crucial for the LAG3-mediated suppression of antitumor immunity in vivo. Consistently, LAG3 therapeutic antibodies repress LAG3 ubiquitination, correlating with their checkpoint blockade effects. Moreover, patient cohort analyses suggest that LAG3/CBL coexpression could serve as a biomarker for response to LAG3 blockade. Collectively, our study reveals an immune-checkpoint-triggering mechanism with translational potential in cancer immunotherapy.