The ligand-dependent suppression of TCR signaling by the immune checkpoint receptor LAG3 depends on the cytoplasmic RRFSALE motif.

Lymphocyte activation gene 3 (LAG3) is an inhibitory immune checkpoint receptor that restrains autoimmune and antitumor responses, but its evolutionarily conserved cytoplasmic tail lacks classical inhibitory motifs. Major histocompatibility complex class II (MHC class II) is an established LAG3 ligand, and fibrinogen-like protein 1 (FGL1), lymph node sinusoidal endothelial cell C-type lectin (LSECtin), and Galectin-3 have been proposed as alternative binding partners that play important roles in LAG3 function. Here, we used a fluorescent human T cell reporter system to study the function of LAG3. We found that LAG3 reduced the response to T cell receptor stimulation in the presence of MHC class II molecules to a lesser extent compared with the receptor programmed cell death protein 1. Analysis of deletion mutants demonstrated that the RRFSALE motif in the cytoplasmic tail of LAG3 was necessary and sufficient for LAG3-mediated inhibition. In this system, FGL1, but not LSECtin or Galectin-3, acted as a LAG3 ligand that weakly induced inhibition. LAG3-blocking antibodies attenuated LAG3-mediated inhibition in our reporter cells and enhanced reporter cell activation even in the absence of LAG3 ligands, indicating that they could potentially enhance T cell responses independently of their blocking effect. Editor's summary: Despite lacking classical inhibitory domains, the immune checkpoint receptor LAG3 inhibits T cell receptor (TCR) signaling when it is engaged by MHC class II molecules or other ligands. The LAG3-blocking antibody relatlimab is used clinically in combinatorial immunotherapy for melanoma. Aigner-Radakovics et al. uncovered a motif in the cytoplasmic tail of LAG3 that was necessary and sufficient for ligand-dependent, LAG3-mediated inhibition of TCR signaling in a human T cell reporter system. A LAG3-blocking antibody related to relatlimab boosted TCR signaling whether or not LAG3 ligands were present, suggesting that such antibodies may enhance antitumor immunity independently of ligand activation of LAG3. —Annalisa M. VanHook [ABSTRACT FROM AUTHOR]