Understanding LAG-3 Signaling
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International Journal of Molecular Sciences Review Understanding LAG-3 Signaling Luisa Chocarro 1 , Ester Blanco 1 , Miren Zuazo 1, Hugo Arasanz 1,2 , Ana Bocanegra 1, Leticia Fernández-Rubio 1, Pilar Morente 1, Gonzalo Fernández-Hinojal 1,2, Miriam Echaide 1, Maider Garnica 1, Pablo Ramos 1, Ruth Vera 2, Grazyna Kochan 1,* and David Escors 1,* 1 Oncoimmunology Group, Navarrabiomed-Public University of Navarre, IdISNA, 31008 Pamplona, Navarra, Spain; [email protected] (L.C.); [email protected] (E.B.); [email protected] (M.Z.); [email protected] (H.A.); [email protected] (A.B.); [email protected] (L.F.-R.); [email protected] (P.M.); [email protected] (G.F.-H.); [email protected] (M.E.); [email protected] (M.G.); [email protected] (P.R.) 2 Department of Medical Oncology, Complejo Hospitalario de Navarra CHN-IdISNA, 31008 Pamplona, Navarra, Spain; [email protected] * Correspondence: [email protected] (G.K.); [email protected] (D.E.) Abstract: Lymphocyte activation gene 3 (LAG-3) is a cell surface inhibitory receptor with multiple biological activities over T cell activation and effector functions. LAG-3 plays a regulatory role in immunity and emerged some time ago as an inhibitory immune checkpoint molecule comparable to PD-1 and CTLA-4 and a potential target for enhancing anti-cancer immune responses. LAG-3 is the third inhibitory receptor to be exploited in human anti-cancer immunotherapies, and it is considered a potential next-generation cancer immunotherapy target in human therapy, right next to PD-1 and CTLA-4. Unlike PD-1 and CTLA-4, the exact mechanisms of action of LAG-3 and its relationship with other immune checkpoint molecules remain poorly understood. This is partly caused by the Citation: Chocarro, L.; Blanco, E.; presence of non-conventional signaling motifs in its intracellular domain that are different from other Zuazo, M.; Arasanz, H.; Bocanegra, conventional immunoregulatory signaling motifs but with similar inhibitory activities. Here we A.; Fernández-Rubio, L.; Morente, P.; summarize the current understanding of LAG-3 signaling and its role in LAG-3 functions, from its Fernández-Hinojal, G.; Echaide, M.; mechanisms of action to clinical applications. Garnica, M.; et al. Understanding LAG-3 Signaling. Int. J. Mol. Sci. 2021, Keywords: LAG-3; immune checkpoint; cancer signaling; immunotherapy; targeted therapy 22, 5282. https://doi.org/10.3390/ ijms22105282 Received: 22 April 2021 1. Molecular Characterization of LAG-3 Accepted: 13 May 2021 1.1. Function Published: 17 May 2021 LAG-3 (CD223) is an inhibitory receptor first described in in vitro-activated T cells. LAG-3 has multiple biological effects on the function of T cells and CD4 T cell activation, Publisher’s Note: MDPI stays neutral usually inhibitory [1,2]. LAG-3 does not seem to inhibit T cell function in the absence with regard to jurisdictional claims in published maps and institutional affil- of CD4 activation but may interfere with CD4 coreceptor function. LAG-3 negatively iations. regulates proliferation, activation, effector function, and homeostasis of both CD8 and CD4 T cells, as shown in LAG-3 knockout mice and by LAG-3 blockade with antibodies in human cells [1,3–5]. Thus, the evidence suggests that LAG-3 interferes with a common pathway to both CD4 and CD8 activation and regulates the activation and expansion of T memory cells [6]. Copyright: © 2021 by the authors. LAG-3 plays a regulatory role in the immune system comparable to PD-1 and CTLA-4, Licensee MDPI, Basel, Switzerland. generally consisting of inhibition of cell proliferation, immune function, cytokine secretion, This article is an open access article distributed under the terms and and homeostasis [1–3,7]. LAG-3 can be upregulated under various antigen stimulation conditions of the Creative Commons conditions [3,8,9]. Its expression is induced by TCR stimulation or cytokine stimulation, and Attribution (CC BY) license (https:// LAG-3 is upregulated within activated, cytokine-expressing T cells [9–12]. LAG-3 associates creativecommons.org/licenses/by/ with the TCR:CD3 complex at the T cell membrane and negatively regulates TCR signal 4.0/). transduction, which in turn terminates cell proliferation and cytokine secretion in response Int. J. Mol. Sci. 2021, 22, 5282. https://doi.org/10.3390/ijms22105282 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5282 2 of 20 Int. J. Mol. Sci. 2021, 22, 5282 2 of 20 TCR signal transduction, which in turn terminates cell proliferation and cytokine secre- tion in response to CD3 signaling [13]. LAG-3 and CD3 co-engagement in the immuno- logical synapse tois necessary CD3 signaling to down-modul [13]. LAG-3ate and TCR CD3 signal co-engagement transduction. in theAmong immunological other synapse is consequences, thenecessary simultaneous to down-modulate engagement TCRof LAG-3/TCR signal transduction. with their Amongligands otherinhibits consequences, the TCR:CD3-dependentsimultaneous intracellular engagement calcium offlux LAG-3/TCRes. Similarly with to theirPD-1, ligands constitutive inhibits LAG-3 TCR:CD3-dependent expression is frequentlyintracellular associated calcium with fluxes. exhaus Similarlyted T cells, to PD-1, and constitutive it is generally LAG-3 regarded expression as is frequently an exhaustion markerassociated for CD4 with and exhausted CD8 T Tcells cells, in andresponse it is generally to repetitive regarded antigen as anstimula- exhaustion marker for tion in cancer andCD4 chronic and CD8viral Tinfections cells in response [14–19]. Indeed, to repetitive LAG-3 antigen has been stimulation found to inphys- cancer and chronic ically associate withviral TCR infections in both [14 CD8–19 ].and Indeed, CD4 T LAG-3 cells following has been foundTCR engagement, to physically down- associate with TCR in regulating TCR-dependentboth CD8 and signaling CD4 T cellscascades following and thereby TCR engagement, dampening downregulatingT cell responsesTCR-dependent [13] (Figure 1). signaling cascades and thereby dampening T cell responses [13] (Figure1). Figure 1. SchematicFigure 1. Schematic representation representation of the molecular of the molecular interactions interactions occurring occurring within the within immunological the immunologi- synapse between a T cell andcal an synapse antigen-presenting between a T cell cell (APC) and an during antigen-presen antigen presentationting cell (APC) and during T cell an activation.tigen presentation The TCR/CD3 and and MHC T cell activation. The TCR/CD3 and MHC complexes are highlighted in yellow. Some of the well- complexes are highlighted in yellow. Some of the well-known co-stimulatory and co-inhibitory receptor–ligand interactions known co-stimulatory and co-inhibitory receptor–ligand interactions are shown in the figure, linked are shown in the figure, linked by red lines for activating interactions and with blue lines for inhibitory interactions. by red lines for activating interactions and with blue lines for inhibitory interactions. Antigenic pep- Antigenictides peptides presented presented by APCs by APCs loaded loaded in MHC inMHC molecules molecules are specifically are specifically recognized recognized by the by TCR, the while TCR, while other interactionsother take interactions place simultaneously take place to simultaneously deliver co-stimulatory to deliver and co-stimulatory co-inhibitory signals. and co-inhibitory These interactions signals. are integrated within T cells.These The interactions LAG-3 molecule are integrated is highlighted within T in cells blue.. The LAG-3 molecule is highlighted in blue. 1.2. Genetic Structure1.2. Geneticof the LAG-3 Structure Gene of and the Domain LAG-3 Gene Organization and Domain Organization Back in 1990, Dr.Back Frédéric in 1990, Triebel Dr. Fr andédé colleaguesric Triebel and identified colleagues lag-3 identified as a novellag-3 lympho-as a novel lymphocyte cyte activation geneactivation closely gene related closely to CD4. related The to authors CD4. The found authors a 2-kb found mRNA a 2-kb to be mRNA selec- to be selectively tively transcribedtranscribed in activated in activatedhuman T human and NK T andcells NK [8]. cells Lag-3 [8 ].encodesLag-3 encodes a protein a protein with a with a molecular molecular weightweight of 70 kDa of 70 and kDa resides and resides on the on distal the distal part partof the of short the short arm arm of chromosome of chromosome 12 (12p13.32) 12 (12p13.32) in inhumans humans and and in in chromosome chromosome 6 6in in mouse. mouse. The The lag-3lag-3 genegene encodes encodes a 498- 498-amino acid type amino acid type II membrane protein [20] [20].. Its Its locus locus isis adjacent adjacent to tothat that of ofthe the CD4 CD4 co-recep- co-receptor and has a tor and has a similarsimilar sequence sequence and and intron/exo intron/exonn organization, organization, strongly strongly indicating indicating that lag- that lag-3 and CD4 genes have evolved by gene duplication from a pre-existing common evolutionary ancestor gene encoding a two-IgSF-domain structure. Int. J. Mol. Sci. 2021, 22, 5282 3 of 20 Int. J. Mol. Sci. 2021, 22, 5282 3 of 20 3 and CD4 genes have evolved by gene duplication from a pre-existing common evolu- tionary ancestor gene encoding a two-IgSF-domain structure. The LAG-3 protein structure can be divided into extracellular, transmembrane,transmembrane, and intracellular regions regions (Figure (Figure 2).2). The The gene gene has has 8 8exons.