LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4+ and CD8+ T cells, in the absence of binding to major histocompatibility complex class II—its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid–proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor–TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies.
Your institute does not have access to this article
Subscription info for Chinese customers
We have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data generated or analyzed during this study are included in this published article and its supplementary information files. Source data are provided with this paper.
Andrews, L. P., Marciscano, A. E., Drake, C. G. & Vignali, D. A. LAG3 (CD223) as a cancer immunotherapy target. Immunol. Rev. 276, 80–96 (2017).
Maruhashi, T., Sugiura, D., Okazaki, I. M. & Okazaki, T. LAG-3: from molecular functions to clinical applications. J. Immunother. Cancer 8, e001014 (2020).
Ruffo, E., Wu, R. C., Bruno, T. C., Workman, C. J. & Vignali, D. A. A. Lymphocyte-activation gene 3 (LAG3): the next immune checkpoint receptor. Semin. Immunol. 42, 101305 (2019).
Huard, B. et al. Characterization of the major histocompatibility complex class II binding site on LAG-3 protein. Proc. Natl Acad. Sci. USA 94, 5744–5749 (1997).
Workman, C. J. & Vignali, D. A. Negative regulation of T cell homeostasis by lymphocyte activation gene-3 (CD223). J. Immunol. 174, 688–695 (2005).
Workman, C. J. & Vignali, D. A. The CD4-related molecule, LAG-3 (CD223), regulates the expansion of activated T cells. Eur. J. Immunol. 33, 970–979 (2003).
Woo, S. R. et al. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res. 72, 917–927 (2012).
Blackburn, S. D. et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat. Immunol. 10, 29–37 (2009).
Wherry, E. J. T cell exhaustion. Nat. Immunol. 12, 492–499 (2011).
Workman, C. J., Dugger, K. J. & Vignali, D. A. Cutting edge: molecular analysis of the negative regulatory function of lymphocyte activation gene-3. J. Immunol. 169, 5392–5395 (2002).
Maeda, T. K., Sugiura, D., Okazaki, I. M., Maruhashi, T. & Okazaki, T. Atypical motifs in the cytoplasmic region of the inhibitory immune co-receptor LAG-3 inhibit T cell activation. J. Biol. Chem. 294, 6017–6026 (2019).
Baixeras, E. et al. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J. Exp. Med. 176, 327–337 (1992).
Grebinoski, S. & Vignali, D. A. Inhibitory receptor agonists: the future of autoimmune disease therapeutics? Curr. Opin. Immunol. 67, 1–9 (2020).
Szent-Gyorgyi, C. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26, 235–240 (2008).
Perkins, L. A. et al. High-content surface and total expression siRNA kinase library screen with VX-809 treatment reveals kinase targets that enhance F508del-CFTR rescue. Mol. Pharm. 15, 759–767 (2018).
Samir, P. et al. DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome. Nature 573, 590–594 (2019).
Fu, G. et al. Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine. Nature 595, 724–729 (2021).
Chen, F., Tillberg, P. W. & Boyden, E. S. Optical imaging. Expansion microscopy. Science 347, 543–548 (2015).
Wassie, A. T., Zhao, Y. & Boyden, E. S. Expansion microscopy: principles and uses in biological research. Nat. Methods 16, 33–41 (2019).
Huang, W. Y. C., Ditlev, J. A., Chiang, H. K., Rosen, M. K. & Groves, J. T. Allosteric modulation of Grb2 recruitment to the intrinsically disordered scaffold protein, LAT, by remote site phosphorylation. J. Am. Chem. Soc. 139, 18009–18015 (2017).
Bettini, M. et al. Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3. J. Immunol. 187, 3493–3498 (2011).
Huang, C. T. et al. Role of LAG-3 in regulatory T cells. Immunity 21, 503–513 (2004).
Woo, S. R. et al. Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4. Eur. J. Immunol. 40, 1768–1777 (2010).
Workman, C. J. et al. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J. Immunol. 172, 5450–5455 (2004).
Grosso, J. F. et al. Functionally distinct LAG-3 and PD-1 subsets on activated and chronically stimulated CD8 T cells. J. Immunol. 182, 6659–6669 (2009).
Grosso, J. F. et al. LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems. J. Clin. Invest. 117, 3383–3392 (2007).
Veillette, A., Bookman, M. A., Horak, E. M. & Bolen, J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase Lck. Cell 55, 301–308 (1988).
Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L. & Schlossman, S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc. Natl Acad. Sci. USA 85, 5190–5194 (1988).
Horkova, V. et al. Dynamics of the coreceptor-LCK interactions during T cell development shape the self-reactivity of peripheral CD4 and CD8 T cells. Cell Rep. 30, 1504–1514 e1507 (2020).
Kim, P. W., Sun, Z. Y., Blacklow, S. C., Wagner, G. & Eck, M. J. A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8. Science 301, 1725–1728 (2003).
Alberts, I. L., Nadassy, K. & Wodak, S. J. Analysis of zinc binding sites in protein crystal structures. Protein Sci. 7, 1700–1716 (1998).
Rigo, A. et al. Interaction of copper with cysteine: stability of cuprous complexes and catalytic role of cupric ions in anaerobic thiol oxidation. J. Inorg. Biochem. 98, 1495–1501 (2004).
Lee, K. H. et al. C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167, 774–788 e717 (2016).
Mitrea, D. M. & Kriwacki, R. W. Phase separation in biology; functional organization of a higher order. Cell Commun. Signal 14, 1 (2016).
Storch, S., Pohl, S. & Braulke, T. A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting. J. Biol. Chem. 279, 53625–53634 (2004).
Johnson, A. O., Lampson, M. A. & McGraw, T. E. A di-leucine sequence and a cluster of acidic amino acids are required for dynamic retention in the endosomal recycling compartment of fibroblasts. Mol. Biol. Cell 12, 367–381 (2001).
Uversky, V. N. The alphabet of intrinsic disorder: II. Various roles of glutamic acid in ordered and intrinsically disordered proteins. Intrinsically Disord. Proteins 1, e24684 (2013).
Miyazaki, T., Dierich, A., Benoist, C. & Mathis, D. Independent modes of natural killing distinguished in mice lacking Lag3. Science 272, 405–408 (1996).
Kaye, J. et al. Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor. Nature 341, 746–749 (1989).
Singh, S. K. et al. Mapping the interaction between the cytoplasmic domains of HIV-1 viral protein U and human CD4 with NMR spectroscopy. FEBS J. 279, 3705–3714 (2012).
Huppa, J. B. et al. TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010).
Kaizuka, Y., Douglass, A. D., Varma, R., Dustin, M. L. & Vale, R. D. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proc. Natl Acad. Sci. USA 104, 20296–20301 (2007).
Liedmann, S. et al. Viral suppressors of the RIG-I-mediated interferon response are pre-packaged in influenza virions. Nat. Commun. 5, 5645 (2014).
Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).
Keller, R. The Computer Aided Resonance Assignment 1st edn (CANTINA, 2004).
Santoro, M. M. & Bolen, D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27, 8063–8068 (1988).
We wish to thank everyone in the Vignali laboratory (Vignali-lab.com; @Vignali_Lab) for all their constructive comments and advice during this project. The authors would like to thank G. Lennon, R. Cross and P. Ingle of the St. Jude Immunology Flow Lab for cell sorting and valuable assistance; A. Yates, D. Falkner and H. Shen from the Immunology Flow Core at the University of Pittsburgh for cell sorting; A. McKenna and K. Forbes for maintenance, breeding and genotyping of mouse colonies at St. Jude Children’s Research Hospital; E. Brunazzi for maintenance, breeding and genotyping of mouse colonies at the University of Pittsburgh; and the staffs of the Shared Animal Resource Center and the Division of Laboratory Animal Services for the animal husbandry A. Philips for assistance with NMR data analysis. Images were acquired, in part, at the Cell and Tissue Imaging Center, and peptide synthesis was performed by the Hartwell Center for Macromolecular Synthesis, which are supported by SJCRH and NCI P30 CA021765. This work was supported by the National Institutes of Health (P01 AI108545, R01 AI129893 to D.A.A.V.; R01 AI144422 to D.A.A.V and C.J.W.), NCI Comprehensive Cancer Center Support CORE grant (CA047904, to D.A.A.V. and R.K.), and ALSAC (to D.A.A.V. and R.K.).
D.M.M. is an employee and stock holder of Dewpoint Therapeutics. D.A.A.V. and C.J.W. have patents covering LAG3, with others pending, and are entitled to a share in net income generated from licensing of these patent rights for commercial development. D.A.A.V. is a cofounder and stock holder at Novasenta, Potenza, Tizona, Trishula; is a stock holder at Oncorus, Werewolf, Apeximmune; has patents licensed and royalties at Astellas, BMS, Novasenta; is a scientific advisory board member at Tizona, Werewolf, F-Star, Bicara, Apeximmune, T7/Imreg Bio; is a consultant at Astellas, BMS, Almirall, Incyte, G1 Therapeutics; has received research funding at BMS, Astellas and Novasenta. The remaining authors declare no competing interests.
Peer review information
Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Ioana Visan, in collaboration with the Nature Immunology team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
(a) Thymidine incorporation proliferation assay with purified CD4+ T cells from spleen and lymph nodes of Lag3+/+ and Lag3–/– mice stimulated with plate-bound CD3ε and CD28 Abs for 72 h in the absence (a) or presence of isotype control or anti-LAG3 blocking antibodies (b). (c) LAG3 expression of Lag3+/+ CD4+ T cells, isolated as above following activation with CD3ε and CD28 Abs at 48 hr. Cells were gated on a live lymphocyte gate, and CD4. (d) Diagram depicting TIRF microscopy analysis on stimulating lipid bilayer. (e) Western blot analysis and quantification of TCR–proximal signaling events in Lag3+/+ or Lag3–/– CD4+ T cells stimulated for 48 hr with CD3ε and CD28 Abs to induce LAG3 expression, allowed to rest in the presence of IL-2 and then restimulated for 5, 15 and 30 min with anti-CD3. Quantitation determined by percent density of Lag3–/– compared to Lag3+/+. Data in (a) represents the mean of n = 16 (Lag3+/+) and n = 16 (Lag3–/–) individual mice. Data in (b) represents the mean of n = 6 (Lag3+/+) and n = 6 (Lag3–/–) individual mice. Data in (e) represents the mean of 4-8 individual experiments. Statistical analysis performed using Student’s unpaired two-sided t test with P values noted in figures.
(a) Diagram depicting TIRF microscopy analysis of Lag3–/– CD4+ T cells containing FAP-tagged LAG3 in the presences of MGnBu to visualize the FAP, stimulated on a planar lipid bilayer containing ICAM and biotinylated TCRβ Ab, with streptavidin Alexa 488 to allow for visualization of TCR clustering. (b) Real-time fluorescent TIRFM visualizing LAG3 (red) and TCR (green) of CD8+ T cells, isolated from spleen and lymph nodes of Lag3–/–mice, containing FAP-tagged LAG3 stimulated on a planar lipid bilayer containing TCRβ Ab for 15 minutes (Scale bar = 5 µm). (c) Activated T cells were immunolabeled for Tubulin (green) and mitochondria (Red) and imaged using 3-dimensional super-resolution confocal either pre- (upper) or post-expansion microscopy (lower) and their maximum cell width determined as a measure of fold expansion. (d) Lag3+/+ and Lag3–/– CD4+ and CD8+ T cells, isolated as above, were immunolabeled to detect TCR and LAG3, and subjected to expansion microscopy with the degree of colocalization presented (Scale bar = 10 µm). (e) Coimmunoprecipitation of the TCR–CD3 complex with LAG3 in resting Lag3+/+ and Lag3–/– transgenic CD4+ T cells and stimulated in the presence of peptide and MHC class II. (f) Molecular distribution of TCR and LAG3 in non-stimulated Lag3+/+ CD4+ and CD8+ T cells, isolated as above, as determined by STORM with quantification shown on the right (Scale bar = 1 µm). Data in (b) and (d) is representative of 3 and 2 independent experiments respectively. Statistics (d, f) was determined by Wilcoxon matched pairs signed rank test or student’s unpaired two-sided t test (c) with P values noted in figures.
(a) Dynamic association of LAG3, TCR and CD8 on Lag3+/+ CD8+ T cells stimulated with TCRβ Ab using sensitized emission FRETc with quantification shown (Scale bar = 5 µm). (b) The average number of LAG3 and CD4 molecules that are within 25 nm x 25 nm x 50 nm area around the TCR in Lag3+/+ CD4+ T cells, isolated as above, following stimulation with TCRβ Ab as determined by the STORM-derived coordinates. Data are representative of at least 3–5 experiments. (c) Diagram depicting a fluid planar lipid bilayer system for analysis of protein–protein interactions. In the system, fluorescently tagged (TAMRA) CD4CT or CD8CT are anchored to the lipid bilayer via his-binding phospholipids resulting in fluid, randomly distributed TAMRA molecules visualized using confocal microscopy. Protein interactions are observed following addition of a peptide (LAG3CT) resulting in phase separation and redistribution of molecules into supramolecular clusters. (d, e) Molecular distribution of CD4 (d), CD8 (e) and Lck within the IS of Lag3+/+ and Lag3–/– CD4+ or CD8+ T cells, isolated as above, as determined by STORM with quantification of the Colocalization index (Scale bar = 0.5 µm). STORM imaging data are representative of 15 data sets derived from 3 separate experiments. Statistics determined by unpaired Student’s two sided t test (a, b) and by Wilcoxon matched pairs signed rank test (d, f). P values are noted in figures.
Sequence alignment of LAG3CT with acidic residues highlighted red.
(a) LAG3CT ‘EP’ and mutant peptide sequences. (b) Diagram depicting a fluid planar lipid bilayer system for analysis of the association/disassociation of p56lck from CD4 or CD8. In the system, TAMRA-labeled p56lck is associated with CD4CT or CD8CT anchored to the lipid bilayer via his-binding phospholipids resulting in fluid, randomly distributed TAMRA molecules visualized using confocal microscopy. Dissociation of p56lck is observed following addition of a peptide (LAG3CT) resulting in a reduction of TAMRA-labeled p56lck and reduced MFI. (c) Determination of phase separation and intermolecular interactions using TAMRA-labeled membrane-tethered CD4CT with AF647-labeled LAG3CT or LAG3CT-QP mutant as photoquencher. Quantification of CD4 quenching or recovery post-photobleaching of acceptor is shown from 2 independent experiments (Scale bar = 1 µm). (d) hCD4 and hCD8 CT sequence. Statistics determined by unpaired Student’s two sided t test. P values are noted in figures.
(a) The disordered cytoplasmic tail of hLAG3 binds Zn2+ weakly, in an entropically driven thermodynamic process. ITC curves at 15°C of Zn2+ titrated into 75 μM hLAG3 peptides, in 10 mM Tris pH 7.0 buffer show that acidic residues are required for metal binding. (b) Cytoplasmic tails of hLck and hCD4 fold upon binding in the presence of Zn2+ as described in Kim PW et al., Science (2003). 1H/15N HSQC spectra of a 50 μM 1:1 complex 15N-Lck: CD4, in the presence of 1.2 molar equivalents of Zn2+ (blue) and the same sample treated with 0.25 mM EDTA (red).
(a, b) Analysis of TCR-induced signaling events in Lag3–/–CD8+ T cells, isolated as above, transduced with LAG3WT or LAG3 functional domain mutants and stimulated with TCRβ Abs. Representative super-resolution confocal images are depicted, with quantification of single cell intensity measurements collected from two independent experiments presented as percent of LAG3-deficient parental cells as control (Scale bar 2 µm). Statistics determined by unpaired Student’s two sided t test. P values are noted in figures.
Supplementary Tables 1–3 and Videos 1–3.
TIRFM visualizing and TCR (green) of Lag3–/– CD4+ T cells containing FAP-tagged LAG3 stimulated on a planar lipid bilayer with TCRβ Ab for 15 min. Scale bar, 5 µm.
TIRFM visualizing LAG3 (red) of Lag3–/– CD4+ T cells isolated as above, containing FAP-tagged LAG3 stimulated on a planar lipid bilayer with TCRβ Ab for 15 min. Scale bar, 5 µm.
TIRFM visualizing both LAG3 (red) and TCR (green) of Lag3–/– CD4+ T cells isolated as above, containing FAP-tagged LAG3 stimulated on a planar lipid bilayer with TCRβ Ab for 15 min. Scale bar, 5 µm.
About this article
Cite this article
Guy, C., Mitrea, D.M., Chou, PC. et al. LAG3 associates with TCR–CD3 complexes and suppresses signaling by driving co-receptor–Lck dissociation. Nat Immunol 23, 757–767 (2022). https://doi.org/10.1038/s41590-022-01176-4
Nature Immunology (2022)
Nature Immunology (2022)
Nature Reviews Drug Discovery (2022)