Dissociation of LAG-3 inhibitory cluster from TCR microcluster by immune checkpoint blockade

Lymphocyte activation gene (Lag)-3 is an inhibitory co-receptor and target of immune checkpoint inhibitor (ICI) therapy for Cancer. The dynamic behavior of LAG-3 was analyzed at the immune synapse upon T-cell activation to elucidate the LAG-3 inhibitory mechanism. LAG-3 formed clusters and co-localized with T-cell receptor microcluster (TCR-MC) upon T-cell activation similar to PD-1. LAG-3 blocking antibodies (Abs) inhibited the co-localization between LAG-3 and TCR-MC without inhibiting LAG-3 cluster formation. LAG-3 also inhibited MHC-II-independent stimulation and LAG-3 Ab, which did not block MHC-II binding could still block Lag-3's inhibitory function, suggesting that the LAG-3 Ab blocks the LAG-3 inhibitory signal by dissociating the co-assembly of TCR-MC and LAG-3 clusters. Consistent with the combination benefit of PD-1 and LAG-3 Abs to augment T-cell responses, bispecific LAG-3/PD-1 antagonists effectively inhibited both cluster formation and co-localization of PD-1 and LAG-3 with TCR-MC. Therefore, LAG-3 inhibits T-cell activation at TCR-MC, and the target of LAG-3 ICI is to dissociate the co-localization of LAG-3 with TCR-MC.