(868) Mechanisms Underlying LAG3 Immune Checkpoint Function

Introduction/Rationale: Immune checkpoint blockade was recognized with the 2018 Nobel Prize in Physiology or Medicine for its revolutionary impact on cancer therapy. LAG3 has emerged as a promising target in cancer immunotherapy, with the FDA approval of relatlimab establishing it as the third immune checkpoint blockade with demonstrated clinical efficacy. However, the molecular mechanisms underlying LAG3-mediated T cell inhibition remain incompletely understood.

Methods: We employed a multidisciplinary strategy to investigate LAG3 signaling, integrating site-directed mutagenesis, biochemical assays, and mass spectrometry. The functional consequences of LAG3 signaling were assessed through T cell proliferation and ELISA measurement of IL-2, while membrane binding properties were evaluated using FRET and NMR. Finally, these mechanistic insights were validated in a clinical context through analysis of single-cell RNA sequencing (scRNA-seq) and immunohistochemistry (IHC) data from patient cohorts treated with PD-1/LAG3 dual blockade.

Results: OUR RECENT STUDIES demonstrated that LAG3 undergoes robust non-K48-linked polyubiquitination upon ligand engagement, which promotes its inhibitory function through disruption of membrane binding by the juxtamembrane basic residue-rich sequence (Cell 2025). Building upon this foundation, our ongoing investigations are uncovering the downstream signaling mechanisms by which LAG3 suppresses T cell function. Through comprehensive proteomic analysis, we have identified novel LAG3-interacting proteins that mediate its immunosuppressive effects. Furthermore, we have discovered additional post-translational modifications on LAG3 that regulate its checkpoint activity in a context-dependent manner.

Conclusion: Collectively, our findings reveal a multi-layered regulatory network controlling LAG3 checkpoint signaling, which may provide new therapeutic opportunities for optimizing LAG3-targeted cancer immunotherapy.