The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible ‘loop 2’ region in LAG3 domain 1. We also defined the LAG3–FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.
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We thank the staff at the 23-ID-D, 22-ID and 19-ID beamlines of the Advanced Photon Source for assistance with remote X-ray data collection. We thank D. Gonzalez-Perez and E. Medina for proofreading and thoughtful comments. The following reagents were obtained through the NIH Tetramer Core Facility: biotinylated MHCII proteins including I-A(b)MOG, I-A(b)CLIP, I-A(d)CLIP, I-A(g7)CLIP and I-E(k)CLIP, HLA-DR4CLIP, HLA-DP2CLIP and HLA-DQ2CLIP. V.C.L., Q.M., S.S. and C.M. are supported by a V Scholar grant from the V Foundation, a Rita Allen Scholars grant and NIH grant no. R35GM133482. G.K.A., C.W. and A.C. are supported by NIH grant no. P01AI120943. B.R. is supported by an NIH/National Cancer Institute grant no. R01CA230610. Support for T.H.T. and shared resources were provided by a Moffitt Cancer Center Support NIH grant no. P30CA076292.
V.C.L. is a consultant on an unrelated project for Cellestia Biotech. The other authors declare no competing interests.
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a, Yeast display selection strategy used to isolate high-affinity FGL1FD binders from the hLAG3D12 mutant library. Flow cytometry dot plots depict the first round (upper panel) and final round of selection (lower panel) stained with 100 nM FGL1FD tetramers and 20 nM FGL1FD monomers, respectively. The gating strategies for yeast and single cells were shown in the bottom. b, Mutation-frequency map generated from sequencing of the five clones isolated from the final round of yeast selection. The recurring mutation, M171I, is highlighted in blue. c, Table of clones containing the M171I mutation. d, Dose-response titrations of yeast expressing hLAG3D12 or LAG3D12 variants with biotinylated FGL1FD. e, SDS-PAGE analysis of LAG3 protein expression and secretion. Two replicated transfections were performed for hLAG3, hLAG3*, hLAG3D12, hLAG3*D12 (hLAG3D12 with M171I mutation). High-Five cells were inoculated with consistent titer of virus. After 48 hours, proteins were retrieved using Ni-NTA. Quantifications of the relative intensity of the bands (right panel) were based on triplicates inoculation.
a, Electron density maps of hLAG3*:F7 complex structure at 3.71 Å. Upper panel: the composite omit map; lower panel: the 2Fo-Fc map from the final round of refinement. The maps were contoured at 0.8 σ. b, Alignment of D3 and D4 domains from the two protomers in the hLAG3 dimer reveals a rotation angle of 161° about the D2-D3 hinge. The two protomers are colored in cyan and grey. c, the M171I mutation and its surrounding residues in hLAG3* structure. M167 in mLAG3 is the equivalent site to M171I. d, Structural comparison of D1 domains from LAG3 (mLAG3) and CD4 (PDB ID: 3T0E). MHCII-binding residues of CD4 D1 are shown as sticks and are clustered in an analogous region to LAG3 Loop2. CD4: pink; mLAG3: green; Loop2: blue.
a, The 2Fo-Fc electron density map, contoured at 1.0 σ, surrounding the mLAG3 dimer interface residue Trp180. b, SPR analyses of hLAG3* binding to HLA-DR4HIV. Biotinylated HLA-DR4HIV was immobilized on a SA chip. hLAG3* protein dilutions starting from 20,000 nM were injected in turn. c,d, SPR sensorgram of recombinant mLAG3 flowed over an SA-chip immobilized with I-A(b)MOG (c) or I-A(b)CLIP (d). e, SPR analyses of LAG3 binding affinity binding to human MHCII allomorphs. MHCII proteins bound to CLIP peptides were biotinylated and immobilized on an SA chip and hLAG3* proteins were flowed over the chip. The curves were then fitted to determine the KD. Mean KD ± s.d. was from two independent replicates. f, Sequence alignment of the D2-dimer interface region from multiple LAG3 orthologs. Residues forming dimer contacts are outlined in red, conserved residues are highlighted in green, and biochemically similar residues are colored in green.
a,b, Untreated HuT 78 cells or HuT 78 cells treated with anti-CD3 (1 ug/ml), anti-CD3 /anti-CD28 (1 ug/ml) or PMA (50 ng/ml) for 48 h and LAG3 expression was analyzed by flow cytometry. c, Flow cytometry analyses of HLA-DR expression on unstimulated or stimulated Hut-78 cells. The cells were stained with anti-HLA-DR (clone L243, BioLegend, 1:100). d, Subcellular localization of LAG-3 and HLA-DR on HuT-78 cells. Confocal fluorescence images displaying nuclei (blue), LAG-3 (green) and HLA-DR (red) in HuT-78 cells, either untreated or treated with anti-CD3/anti-CD28. Representative confocal images for the individual channels and merged images are shown in the zoomed panel. One of three representative experiments is shown. Horizontal bars in (b) represent the mean and statistics was determined by one-way ANOVA with P values noted in the figure.
a–c, SPR sensograms measuring hLAG3 binding to MHCII in the presence of scFv-Fc fusion proteins. d, SPR was used to determine scFv:LAG3 binding affinities. e, Flow cytometry analyses of binding of recombinant HLA-DR4HIV(4 µM) to yeast-displayed CD4 or hLAG3 and its mutants. f,g, A luciferase reporter assay was used to assess the ability of 15011-Fc to inhibit LAG3 in the presence of FGL1. To activate TCR signaling, LAG3+ Jurkat cells were either co-cultured with MHCII-expressing Raji cells (f) or stimulated with anti-CD3 (α-CD3) (g). In f and g, the cells were supplemented with 10 nM FGL1 and an isotype control antibody, 15011-Fc, or F7-Fc (300 nM). The luminescence was measured after incubating the cells overnight. In f and g, the graph represents mean ± SD of three replicates from representative of two independent experiments. All statistics was determined by one-way ANOVA, with P values noted in the figure.
a, Flow chart depicting the selection strategy used to isolate mutations that decreased LAG3 binding to FGL1. In the first two rounds, the hLAG3D12 library was negatively selected against Alexa Fluor 647-labeled streptavidin to remove non-specific binders. In rounds 3 through 5, the indicated concentrations of biotinylated FGL1FD were incubated with the library and negative selections were performed to remove FGL1 binders. b, Yeast expressing LAG3 mutants were stained with MHCII tetramers (200 nM) to determine whether the FGL1 loss-of-binding mutations affected LAG3 binding to MHCII. c, SPR-sensograms recorded following the injection of samples containing fixed concentrations of hLAG3 (800 nM) and varying concentrations 15011.scFv over a chip coated with FGL1FD.
a, Structural alignment of the three copies of FGL1FD (FD-a, FD-b and FD-c) in the crystal asymmetric unit. Different P subdomain loop conformations differences are depicted in the zoom panel. b, Structural alignment of fibrinogen-like domains from FGL1, Ang1 (RMSD = 1.12 Å) and FIBCD1 (RMSD = 1.21 Å). The FD-b protomer from the FGL1FD structure was used in this alignment. FGL1, blue; Ang1, yellow; FICD1, gray. c, Grouped residues mutated in the alanine scanning assay. Residues that were mutated in a single construct are colored accordingly. d, Flow cytometry histogram plots depicting increased binding of FGL1FD variant populations following iterative rounds of selection against hLAG3 (left) and a heat map showing the mutation frequency of amino acid substitutions after sequencing six clones (right). e, Yeast expressing hLAG3 were stained with biotinylated FGL1FD and FGL1FD containing the affinity-enhancing G290E mutation (FDG290E).
a, LAG3-NFAT Jurkat T cells were treated with 1, 10 or 100 nM of FGL1 protein and incubated for 30 min. Binding was detected using an anti-LAG3 antibody. Data reflect the mean ± SD of n = 3 technical replicates, with representative of two experiments is shown. b, In the presence of the blocking antibody, 15011Fc, the FGL1-LAG3 clustering effect was diminished. Stacked confocal microscopy images displaying LAG3 (green) and DAPI (blue) in Jurkat T cells expressing LAG3, either untreated or treated with the indicated reagents: 10 nM FGL1, 15011Fc, isotype control, 10 nM FGL1 and 300 nM 15011Fc, or 10 nM FGL1 and 300 nM isotype control. c, The number of LAG3 clusters per cell was quantified and plotted as the average per field of view (FOV) with analyses of n = 7 FOV from one of three representative experiments. d, NFAT luciferase reporter assay performed to assess the effect of FGL1 on LAG3-mediated suppression of T cell activation mediated by anti-CD3. The relative activation was obtained by normalizing baseline-subtracted luciferase signals of tested concentrations to the buffer control group. The graph represents mean ± SD of three independent replicates from one of two representative experiments. All significance was determined by one-way ANOVA, with P values noted in the figure.
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Ming, Q., Celias, D.P., Wu, C. et al. LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition. Nat Immunol 23, 1031–1041 (2022). https://doi.org/10.1038/s41590-022-01238-7
Nature Immunology (2022)