Lag3 Mediates Its Inhibitory Function By Associating With The Tcr:Cd3 Complex And Inducing Co-Receptor:P56(Lck) Dissociation

Abstract Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor that is highly expressed on exhausted T cells in the tumor microenvironment. Despite over ten LAG3-targeting immunotherapeutics in clinical trials, we still do not know how LAG3 mediates its inhibitory activity. We show that LAG3 can function in the absence of MHC class II ligation and that in the absence of LAG3 there is a 2–3 fold increase in T cell receptor signaling resulting in increased proliferation. We show that both murine and human LAG3 molecules track to the immunological synapse (IS) by close association with the T cell receptor (TCR):CD3 complex. A phylogenetically conserved, acidic tandem glutamic acid–proline repeat in the LAG3 cytoplasmic tail can lower local pH at the IS and chelate Zn2+, limiting T cell activating signals by causing the dissociation of CD4:p56lck, which interact via a di-cystine:Zn2+ complex. This in turn causes a loss of co-receptor:TCR signaling. This novel inhibitory pH-mediated, Zn2+-displacement mechanism highlights LAG3 as a “signal disruptor” that can function in an MHC class II independent manner, providing critical insight into the mechanism of action of LAG3-targeting immunotherapies.