{"gene":"LAG3","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2022,"finding":"LAG3 moves to the immunological synapse and associates with the TCR-CD3 complex in CD4+ and CD8+ T cells independently of MHC class II binding. A phylogenetically conserved acidic tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowers pH at the immune synapse and causes dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, resulting in loss of co-receptor-TCR signaling and limited T cell activation.","method":"Co-immunoprecipitation, live-cell imaging, pH-sensing reporters, mutational analysis of cytoplasmic domain, functional T cell activation assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (Co-IP, live imaging, mutagenesis, functional assays) in a single rigorous study establishing a defined molecular mechanism","pmids":["35437325"],"is_preprint":false},{"year":2025,"finding":"LAG3 undergoes robust non-K48-linked polyubiquitination upon engagement of MHC class II or membrane-bound (but not soluble) FGL1. This ubiquitination, mediated redundantly by E3 ligases c-Cbl and Cbl-b, disrupts membrane binding of the juxtamembrane basic residue-rich sequence, stabilizing the LAG3 cytoplasmic tail in a membrane-dissociated conformation that enables inhibitory signaling. LAG3 ubiquitination is required for suppression of antitumor immunity in vivo, and therapeutic antibodies repress LAG3 ubiquitination.","method":"Ubiquitination assays, E3 ligase identification (c-Cbl/Cbl-b), mutagenesis of juxtamembrane domain, in vivo tumor models, patient cohort analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reconstitution-level biochemistry with mutagenesis, E3 ligase identification, and in vivo validation in a single rigorous study","pmids":["40101708"],"is_preprint":false},{"year":2025,"finding":"LAG3's spatial proximity to the TCR (but not CD4 co-receptor), facilitated by cognate peptide-MHC class II, is required for optimal CD4+ T cell suppression. LAG3 forms condensates with TCR signaling component CD3ε through its intracellular FSAL motif, disrupting CD3ε/Lck association. MHC class II interaction alone is insufficient for optimal LAG3 function.","method":"Proximity ligation assays, super-resolution microscopy, co-immunoprecipitation, FSAL-motif mutagenesis, bispecific antibody rescue experiments, autoimmune mouse models","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (structural proximity, Co-IP, mutagenesis, in vivo rescue) in a single rigorous study","pmids":["40592325"],"is_preprint":false},{"year":2022,"finding":"LAG3 dimerization is required for its association with the TCR/CD3 complex and for optimal inhibitory function. A LAG3 mutant unable to dimerize shows perturbed TCR/CD3 association in CD8+ T cells and reduced inhibition in a tumor model. The therapeutic antibody C9B7W disrupts LAG3 dimerization and its TCR/CD3 association without blocking MHC class II binding.","method":"Dimerization-defective LAG3 mutant, co-immunoprecipitation with TCR/CD3, in vivo B16-gp100 tumor model, antibody epitope mapping","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, loss-of-function mutant, and in vivo functional validation in one study","pmids":["38775415"],"is_preprint":false},{"year":2022,"finding":"Crystal and cryo-EM structures of human and murine LAG3 ectodomains reveal a dimeric assembly mediated by Ig domain 2. A flexible 'loop 2' region in LAG3 domain 1 is the epitope for a potent antagonist antibody that blocks both MHC class II and FGL1 interactions. FGL1 cross-linking induces higher-order LAG3 oligomers, implicating ligand-mediated clustering as a mechanism for disrupting T cell activation.","method":"X-ray crystallography, cryo-EM, mutational mapping of LAG3-FGL1 and LAG3-MHC class II interfaces, oligomerization assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure and cryo-EM with mutational validation and functional interface mapping","pmids":["35761082"],"is_preprint":false},{"year":2018,"finding":"Fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent of MHC class II. FGL1 inhibits antigen-specific T cell activation, and blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity in mouse models.","method":"Binding assays, LAG-3/FGL1 blocking monoclonal antibodies, FGL1 knockout mice, in vivo tumor models, T cell activation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding, KO mice, in vivo tumor models, antibody blockade) replicated across systems","pmids":["30580966"],"is_preprint":false},{"year":2022,"finding":"Binding of LAG-3 to stable peptide-MHC class II (pMHCII) complexes, but not to FGL1, induces T cell suppression in vitro. LAG-3 mutants lacking stable pMHCII-binding capacity but not FGL1-binding capacity lost suppressive activity. Targeted disruption of stable pMHCII-binding of LAG-3 in NOD mice recapitulated diabetes exacerbation seen with LAG-3 deficiency, and augmented anti-cancer immunity in C57BL/6 mice, identifying stable pMHCII as the functional ligand for both autoimmunity and anti-cancer immunity.","method":"In vitro T cell suppression assays with LAG-3 binding mutants, knock-in mice with site-specific mutations, autoimmune diabetes model (NOD), tumor models","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution-level in vitro assays with mutagenesis, corroborated by multiple in vivo genetic mouse models","pmids":["35413245"],"is_preprint":false},{"year":2004,"finding":"LAG-3 is proteolytically cleaved within the D4 transmembrane domain connecting peptide, producing a 54-kDa N-terminal extracellular fragment that oligomerizes with full-length LAG-3 (70 kDa) on the cell surface via the D1 domain, and a 16-kDa C-terminal fragment containing the transmembrane and cytoplasmic domains. The 54-kDa fragment is subsequently released as soluble LAG-3 (sLAG-3), a process enhanced by T cell activation in vitro and in vivo.","method":"Biochemical fractionation, Western blotting, domain-deletion mutants, in vivo serum analysis in C57BL/6 and RAG-1−/− mice","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical reconstitution with mutants and in vivo validation in single rigorous study","pmids":["15557174"],"is_preprint":false},{"year":2003,"finding":"LAG-3 function on T cells is mediated via its cytoplasmic domain, and a conserved 'KIEELE' motif within this domain is essential for LAG-3's regulatory activity on T cell expansion.","method":"Retroviral reconstitution of LAG-3 wild-type and cytoplasmic domain mutants in LAG-3−/− T cells, in vitro expansion assays","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis with retroviral reconstitution and functional readout","pmids":["12672063"],"is_preprint":false},{"year":2005,"finding":"LAG-3 negatively regulates T cell homeostasis; LAG-3-deficient mice accumulate twice as many T cells with age, and LAG-3−/− T cells show enhanced homeostatic expansion in lymphopenic hosts. Ectopic expression of wild-type LAG-3, but not a signaling-defective mutant, abrogates this deregulated expansion. Regulatory T cells depend on LAG-3 for optimal suppression of T cell homeostasis.","method":"LAG-3−/− mice, adoptive transfer into lymphopenic hosts, retroviral ectopic expression of wild-type vs. signaling-defective LAG-3, anti-LAG-3 mAb treatment in vivo","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO, adoptive transfer, and mutant reconstitution with consistent results across multiple experiments","pmids":["15634887"],"is_preprint":false},{"year":2004,"finding":"LAG-3 negatively regulates T cell expansion in vivo: LAG-3−/− mice show delayed cell cycle arrest and increased T cell expansion after superantigen stimulation, increased memory T cells after viral infection, and enlarged memory T cell pools. LAG-3 controls the size of the memory T cell pool.","method":"LAG-3−/− mice, superantigen SEB stimulation in vivo, adoptive transfer of LAG-3−/− OT-II T cells, Sendai virus and murine gammaherpesvirus infection models","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — four independent experimental systems in LAG-3−/− mice all showing consistent T cell expansion phenotype","pmids":["15100286"],"is_preprint":false},{"year":2002,"finding":"LAG-3Ig (soluble LAG-3) binds MHC class II molecules in plasma membrane lipid rafts on immature human dendritic cells (DCs) and induces DC maturation: rapid morphological changes, upregulation of costimulatory molecules, IL-12 and TNF-alpha production, and enhanced capacity to stimulate naive T cells for Th1 responses. These effects were not observed with MHC class II-specific mAbs.","method":"LAG-3Ig fusion protein treatment of human monocyte-derived DCs, flow cytometry, cytokine ELISA, T cell co-culture assays, confocal microscopy","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional readouts (morphology, surface markers, cytokines, T cell priming) in a single study","pmids":["11937541"],"is_preprint":false},{"year":2003,"finding":"LAG-3-induced MHC class II signaling in human DCs involves activation of PLCγ2, p72syk, PI3K/Akt, p42/44 ERK, and p38 MAPK pathways, all required for DC maturation. LAG-3 engagement versus anti-MHC class II antibody produces different phosphorylation patterns of c-Akt, indicating qualitative signaling differences dependent on the natural ligand.","method":"Phospho-protein analysis, kinase inhibitor studies, Western blotting in human monocyte-derived DCs, confocal microscopy with soluble LAG-3","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple pathways dissected with specific inhibitors and biochemical readouts in one study","pmids":["12775570"],"is_preprint":false},{"year":2017,"finding":"LAG3 intrinsically limits regulatory T cell (Treg) proliferation and function at inflammatory sites. Treg-specific LAG3 deletion (using Foxp3-Cre) reduces autoimmune diabetes in NOD mice by enhancing Treg proliferation, IL-2-STAT5 signaling, and Eos expression in intra-islet Tregs. LAG3-deficient Tregs outcompete wild-type Tregs specifically at the inflammatory site.","method":"Treg-specific LAG3 conditional knockout (Foxp3-Cre x Lag3-flox), RNA sequencing of intra-islet vs. peripheral Tregs, cotransfer competition experiments, STAT5 phosphorylation assays","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — cell-type-specific KO with transcriptomic analysis and multiple functional readouts","pmids":["28783703"],"is_preprint":false},{"year":2024,"finding":"LAG-3 sustains TOX expression in exhausted CD8 T cells during chronic infection and cancer, maintaining Tex cell durability. LAG-3 controls a CD94/NKG2+ subset of exhausted T cells with enhanced cytotoxicity via recognition of the stress ligand Qa-1b. PD-1 and LAG-3 have distinct non-redundant roles: PD-1 primarily regulates proliferation while LAG-3 primarily regulates effector functions of exhausted T cells.","method":"LAG-3 and PD-1 single/double knockout mice in chronic LCMV infection, flow cytometry, TOX expression analysis, cytotoxicity assays, NKG2/Qa-1b interaction studies","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic dissection with single and double KO mice, multiple functional readouts, and human validation","pmids":["39121847"],"is_preprint":false},{"year":2024,"finding":"Lag3 supports Foxp3+ regulatory T cell suppressive function by restraining Myc-dependent metabolic programming. Lag3 mutation in Tregs increases Myc expression to levels seen in Th1 effector cells, activates PI3K-Akt-Rictor signaling, and dysregulates glycolytic metabolism. Inhibiting PI3K, Rictor, or the Myc target enzyme Ldha restores normal metabolism and suppressive function in Lag3-mutant Tregs.","method":"Treg-specific Lag3 mutant mouse models, RNA sequencing, metabolic profiling, PI3K/Rictor/Ldha inhibitor rescue experiments, in vivo autoimmunity models","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Treg-specific genetic models with transcriptomics, metabolomics, and pharmacological pathway rescue","pmids":["39236718"],"is_preprint":false},{"year":2005,"finding":"LAG-3 co-localizes with CD3, CD4 or CD8 in cholesterol-rich lipid raft aggregations during primary T cell activation. Blocking LAG-3/MHC class II interactions with anti-LAG-3 mAb augments CD69 expression, T cell expansion, cell cycle entry, and Th1 (but not Th2) cytokine production in both CD4+ and CD8+ primary human T cells at low antigen concentrations.","method":"Confocal microscopy for co-localization, anti-LAG-3 blocking mAb, BrdU incorporation, flow cytometry, cytokine ELISA in primary human T cells","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — localization with functional consequence shown, single lab with multiple readouts","pmids":["15885122"],"is_preprint":false},{"year":2015,"finding":"CD4+CD25−LAG3+ regulatory T cells (LAG3+ Tregs) suppress humoral immune responses and B cell activity through TGF-β3 production in an Egr2- and Fas-dependent manner. This suppression requires PD-1 expression on B cells.","method":"In vitro co-culture suppression assays, TGF-β3 neutralization, Egr2-deficient mice, Fas-deficient cells, PD-1-deficient B cells, murine lupus model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mechanism shown with multiple genetic perturbations in one study, single lab","pmids":["25695838"],"is_preprint":false},{"year":2018,"finding":"LAG-3 expressed on Foxp3+ regulatory T cells mediates contact-dependent suppression of CX3CR1+ intestinal macrophages via MHC class II engagement, inhibiting IL-23 and IL-1β production from macrophages and thereby restraining ILC3-mediated IL-22 production and colitis.","method":"Treg-macrophage co-culture contact-dependency assays, LAG-3 blocking antibodies, Treg-specific depletion, colitis mouse model with ILC3 transfer","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-culture and in vivo model with LAG-3 blockade showing defined suppression pathway, single lab","pmids":["30097293"],"is_preprint":false},{"year":2005,"finding":"Soluble LAG-3 (sLAG-3), but not an MHC class II-specific mAb, reduces differentiation of monocytes into macrophages (in GM-CSF) and into dendritic cells (in GM-CSF + IL-4), as shown by decreased CD14 and CD1a expression. DCs differentiated in the presence of sLAG-3 have impaired antigen-presentation capacity.","method":"sLAG-3 treatment of human monocytes, flow cytometry for differentiation markers, T cell proliferation assays","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional assay with soluble protein, single lab, multiple readouts but no mutagenesis or mechanistic dissection","pmids":["15720438"],"is_preprint":false},{"year":2003,"finding":"Soluble LAG-3Ig engagement of MHC class II on immature human DCs induces a distinct chemokine profile: production of IL-8 and MIP-1α/CCL3 (inflammatory), and MDC/CCL22 and TARC/CCL17 (lymph node-homing), with CCR5 downregulation and CCR7 upregulation on DC surface, potentially directing DC migration to lymph nodes.","method":"LAG-3Ig treatment of human monocyte-derived DCs, cytokine ELISA, flow cytometry for chemokine receptors, chemotaxis assays","journal":"Vaccine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional characterization with multiple chemokine readouts, single lab","pmids":["12547595"],"is_preprint":false},{"year":2024,"finding":"Amyloid β precursor-like protein 1 (Aplp1) interacts with Lag3 and facilitates binding, internalization, transmission, and toxicity of pathologic α-synuclein preformed fibrils (PFF). Deletion of both Aplp1 and Lag3 eliminates dopaminergic neuron loss and behavioral deficits induced by α-syn PFF. Anti-Lag3 prevents α-syn PFF internalization by disrupting the Aplp1-Lag3 interaction.","method":"Co-immunoprecipitation of Aplp1-Lag3, double KO mice (Aplp1 and Lag3), α-synuclein PFF injection model, anti-Lag3 antibody blockade, behavioral tests, dopaminergic neuron counts","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus genetic double-KO with functional readout, single lab","pmids":["38821932"],"is_preprint":false},{"year":2021,"finding":"LAG3 is not expressed by neurons in human or murine brains. Overexpression of LAG3 in human neural cells did not worsen α-synuclein pathology ex vivo. LAG3 knockout did not affect survival of A53T α-synuclein transgenic mice or seeded α-synuclein lesions in hippocampal slice cultures, though LAG3 does interact with α-synuclein fibrils with limited specificity.","method":"Immunohistochemistry, RNA-seq, Western blot, LAG3 overexpression in neural cells, LAG3 KO mice, hippocampal slice culture seeding assay, survival analysis","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple negative results with rigorous methods across different model systems consistently showing no neuronal expression or functional role","pmids":["34309222"],"is_preprint":false},{"year":2023,"finding":"LAG-3 expression in microglia is induced by IFN-γ via the STAT1 pathway. Both membrane and soluble forms of LAG-3 are upregulated in IFN-γ-activated microglia. Soluble LAG-3 production is regulated by metalloproteinases ADAM10 and ADAM17. LAG-3 knockdown in microglia promotes nitric oxide production by IFN-γ, indicating LAG-3 restrains microglial activation.","method":"siRNA knockdown of STAT1, metalloproteinase inhibitors (ADAM10/17), IFN-γ stimulation of BV2 and primary microglia, in vivo intracisterna magna IFN-γ injection, nitric oxide assay","journal":"Frontiers in cellular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA knockdown and pharmacological inhibition with functional readout, single lab","pmids":["38026700"],"is_preprint":false},{"year":2018,"finding":"A natural subset of plasma cells distinctively expressing LAG-3 (along with CD200, PD-L1, and PD-L2) produces IL-10 and mediates immune suppression in vivo. These LAG-3+ regulatory plasma cells develop from B cell subsets in a BCR-dependent manner and upregulate IL-10 via a TLR-driven mechanism upon challenge.","method":"Flow cytometry identification of LAG-3+ plasma cell subset, BCR-dependent development assays, TLR stimulation, IL-10 ELISA, in vivo challenge models, transcriptomic and epigenomic profiling","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — cell subset identification with functional in vivo experiments, single lab","pmids":["30005826"],"is_preprint":false}],"current_model":"LAG3 (CD223) is an inhibitory co-receptor that localizes to the immunological synapse, where it associates with the TCR-CD3 complex (requiring LAG3 dimerization and facilitated by cognate pMHC class II proximity) and suppresses T cell activation by: (1) using an acidic glutamic acid-proline repeat in its cytoplasmic tail to lower local pH and dissociate Lck from CD4/CD8 co-receptors; (2) forming condensates with CD3ε via its FSAL motif to disrupt CD3ε/Lck association; and (3) undergoing c-Cbl/Cbl-b-mediated polyubiquitination upon ligand engagement, which releases a juxtamembrane basic sequence from the membrane to permit inhibitory signaling. Its primary functional ligand is stable peptide-MHC class II (pMHCII), with FGL1 serving as an additional ligand that induces LAG3 oligomerization; the LAG3 ectodomain forms a dimer mediated by Ig domain 2 with a flexible loop 2 region in domain 1 engaging both ligands. Beyond T cell-intrinsic signaling, LAG3 on Tregs limits their proliferation and metabolic activity (via restraint of Myc/PI3K-Akt-Rictor signaling) and mediates contact-dependent suppression of antigen-presenting cells through MHC class II; as a soluble form generated by ADAM10/ADAM17-mediated cleavage, it can also induce DC maturation through MHC class II signaling."},"narrative":{"mechanistic_narrative":"LAG3 (CD223) is an inhibitory co-receptor that restrains T cell activation, controls T cell homeostasis and the size of the memory pool, and supports the suppressive function of regulatory T cells [PMID:35437325, PMID:15100286, PMID:28783703]. Upon recruitment to the immunological synapse, LAG3 associates with the TCR-CD3 complex independently of MHC class II, a step requiring LAG3 dimerization, and delivers inhibitory signals through its cytoplasmic tail [PMID:35437325, PMID:38775415]. Mechanistically, an acidic tandem glutamic acid-proline repeat lowers local synapse pH to dissociate Lck from the CD4/CD8 co-receptor, while LAG3 also forms condensates with CD3ε via an intracellular FSAL motif to disrupt CD3ε/Lck association, with cognate peptide-MHC class II proximity to the TCR being required for optimal suppression [PMID:35437325, PMID:40592325]. Ligand engagement triggers c-Cbl/Cbl-b-mediated non-K48 polyubiquitination that releases the membrane-bound juxtamembrane basic sequence, stabilizing the tail in a signaling-competent conformation; this ubiquitination is required for suppression of antitumor immunity [PMID:40101708]. Stable peptide-MHC class II is the functional ligand for both autoimmunity and anti-cancer immunity, with FGL1 serving as an MHC-class-II-independent ligand whose cross-linking drives higher-order LAG3 oligomerization; the ectodomain dimerizes through Ig domain 2, with a domain-1 loop 2 region engaging both ligands [PMID:35761082, PMID:30580966, PMID:35413245]. On Foxp3+ Tregs, LAG3 limits proliferation and metabolic activity by restraining Myc and PI3K-Akt-Rictor signaling and mediates contact-dependent suppression of antigen-presenting cells via MHC class II [PMID:28783703, PMID:39236718, PMID:30097293]. LAG3 is proteolytically cleaved by ADAM10/ADAM17 to release a soluble ectodomain that engages MHC class II on dendritic cells to drive their maturation and signaling [PMID:15557174, PMID:11937541, PMID:38026700].","teleology":[{"year":2002,"claim":"Established that soluble LAG-3 acts as an MHC class II ligand on dendritic cells, defining a function for LAG3 beyond T-cell-intrinsic inhibition.","evidence":"LAG-3Ig fusion protein treatment of human monocyte-derived DCs with morphology, surface marker, cytokine, and T cell priming readouts","pmids":["11937541"],"confidence":"High","gaps":["Did not define the LAG3 cytoplasmic signaling mechanism","Effects shown with soluble fusion protein rather than endogenous cleaved sLAG-3"]},{"year":2003,"claim":"Identified the cytoplasmic determinant of LAG3 inhibitory signaling, showing a conserved KIEELE motif is essential for regulating T cell expansion.","evidence":"Retroviral reconstitution of wild-type and cytoplasmic mutants in LAG-3−/− T cells with expansion assays; parallel DC studies dissecting MHC-class-II signaling pathways","pmids":["12672063","12775570","12547595"],"confidence":"High","gaps":["Downstream effectors of the KIEELE motif not identified at the time","Did not connect motif to TCR-CD3 association"]},{"year":2004,"claim":"Showed LAG-3 negatively regulates T cell expansion and memory pool size in vivo, and that it is proteolytically processed to release a soluble ectodomain.","evidence":"LAG-3−/− mice with superantigen and viral infection models; biochemical fractionation and domain-deletion mapping of cleavage fragments","pmids":["15100286","15557174"],"confidence":"High","gaps":["Protease responsible for cleavage not identified in these studies","Molecular mechanism of inhibition still unresolved"]},{"year":2005,"claim":"Linked LAG3 to homeostatic T cell control and lipid-raft synapse localization, and extended sLAG-3 effects to monocyte differentiation.","evidence":"LAG-3−/− mice with adoptive transfer; confocal co-localization with CD3/CD4/CD8 in lipid rafts; sLAG-3 treatment of human monocytes","pmids":["15634887","15885122","15720438"],"confidence":"High","gaps":["Co-localization data (Medium) not mechanistically dissected","Treg dependence on LAG3 shown functionally but molecular basis unknown"]},{"year":2017,"claim":"Defined a Treg-intrinsic role: LAG3 limits Treg proliferation and function at inflammatory sites by restraining IL-2-STAT5 signaling.","evidence":"Treg-specific conditional knockout (Foxp3-Cre x Lag3-flox), RNA-seq of intra-islet Tregs, competition transfers, STAT5 phosphorylation assays","pmids":["28783703"],"confidence":"High","gaps":["How LAG3 signaling links to STAT5 restraint not resolved","Site-specific competitive advantage mechanism unclear"]},{"year":2018,"claim":"Identified FGL1 as a major MHC-class-II-independent functional ligand and established contact-dependent Treg suppression of APCs through MHC class II.","evidence":"FGL1 binding assays, blocking antibodies, FGL1 KO mice, tumor models; Treg-macrophage co-culture with LAG-3 blockade and colitis model","pmids":["30580966","30097293","30005826"],"confidence":"High","gaps":["Relative contributions of FGL1 versus pMHCII not yet ranked","Receptor conformational consequences of ligand binding unknown"]},{"year":2022,"claim":"Provided the structural and signaling framework: ectodomain dimerization via Ig domain 2, ligand-induced oligomerization, TCR-CD3 association requiring dimerization, and a pH/Lck-dissociation mechanism in the cytoplasmic tail.","evidence":"Crystallography and cryo-EM with interface mutagenesis; dimerization-defective mutants with reciprocal Co-IP and tumor models; Co-IP, live imaging, pH reporters and functional T cell assays","pmids":["35761082","38775415","35437325"],"confidence":"High","gaps":["How dimerization and the acidic E-P repeat are coordinated in time not fully resolved","Stoichiometry of LAG3-TCR-CD3 assembly at the synapse not defined"]},{"year":2022,"claim":"Resolved which ligand drives suppression, showing stable pMHCII binding is the functional ligand for both autoimmunity and anti-cancer immunity.","evidence":"In vitro suppression assays with binding mutants and knock-in mice in NOD diabetes and tumor models","pmids":["35413245"],"confidence":"High","gaps":["Reconciliation with FGL1 ligand activity not fully integrated","Whether different ligands trigger distinct signaling outputs unresolved"]},{"year":2024,"claim":"Distinguished LAG3 from other checkpoints in exhausted T cells and detailed Treg metabolic control by LAG3.","evidence":"LAG-3/PD-1 single and double KO mice in chronic LCMV with TOX and cytotoxicity readouts; Treg-specific Lag3 mutants with transcriptomics, metabolomics, and PI3K/Rictor/Ldha inhibitor rescue","pmids":["39121847","39236718"],"confidence":"High","gaps":["How a single inhibitory receptor selectively shapes effector function versus proliferation not mechanistically defined","Link between membrane signaling and Myc/metabolic restraint not established"]},{"year":2025,"claim":"Completed the proximal signaling model: ligand-induced c-Cbl/Cbl-b ubiquitination releases the juxtamembrane tail for signaling, and CD3ε condensate formation via FSAL disrupts CD3ε/Lck.","evidence":"Ubiquitination assays, E3 ligase identification, juxtamembrane mutagenesis, in vivo tumor models and patient cohorts; proximity ligation, super-resolution imaging, FSAL mutagenesis, bispecific antibody rescue, autoimmune models","pmids":["40101708","40592325"],"confidence":"High","gaps":["Integration of ubiquitination, pH-Lck dissociation, and CD3ε condensation into one ordered pathway not yet formalized","Generality across CD4 versus CD8 T cells partially addressed"]},{"year":2024,"claim":"Explored a non-immune role in neurodegeneration, with conflicting evidence on LAG3 as a mediator of pathologic α-synuclein uptake.","evidence":"Aplp1-Lag3 Co-IP, double-KO mice and anti-Lag3 blockade in α-syn PFF model; contrasted with prior reports of no neuronal LAG3 expression or functional effect, and IFN-γ/STAT1-induced microglial LAG3","pmids":["38821932","34309222","38026700"],"confidence":"Medium","gaps":["Conflicting reports on whether LAG3 contributes to α-synuclein pathology not reconciled","Cell type expressing functionally relevant LAG3 in brain disputed","Mechanism of Aplp1-Lag3 cooperation in fibril internalization incomplete"]},{"year":null,"claim":"How the distinct proximal mechanisms (acidic E-P repeat-driven Lck dissociation, FSAL-CD3ε condensation, Cbl-mediated ubiquitination) are temporally ordered and whether they operate in parallel or sequentially during a single inhibitory event remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified kinetic model linking the three cytoplasmic mechanisms","Relative ligand-specific contributions of pMHCII versus FGL1 to each step not quantified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,8,9]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[6,18]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,7,16]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,6,10,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,12]}],"complexes":[],"partners":["CD3E","FGL1","CBL","CBLB","APLP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P18627","full_name":"Lymphocyte activation gene 3 protein","aliases":[],"length_aa":525,"mass_kda":57.4,"function":"Inhibitory receptor on antigen activated T-cells (PubMed:20421648, PubMed:35761082, PubMed:7805750, PubMed:8647185). Delivers inhibitory signals upon binding to ligands, such as MHC class II, its main ligand present at the surface of antigen-presenting cells (APCs), and FGL1, which is secreted by hepatocytes and certain types of tumor cells (PubMed:30580966, PubMed:32920841, PubMed:35761082, PubMed:39671469, PubMed:7589152, PubMed:8647185, PubMed:9159144). Ligand-binding initiates a signaling that inhibits the T-cell receptor (TCR) in the immunological synapse, preventing T-cell activation (PubMed:40101708). Mechanistically, ligand-binding promotes (1) ubiquitination of the KIEELE motif, unleashing the RRFSALE motif from the membrane and (2) leading to the formation of condensates with the TCR component CD3E, thereby disrupting the association between CD3E and LCK and preventing TCR activation (PubMed:40101708, PubMed:40592325). May inhibit antigen-specific T-cell activation in synergy with PDCD1/PD-1 (By similarity). Negatively regulates the proliferation, activation, effector function and homeostasis of both CD8(+) and CD4(+) T-cells (PubMed:20421648, PubMed:7805750, PubMed:8647185). Also mediates immune tolerance: constitutively expressed on a subset of regulatory T-cells (Tregs) and contributes to their suppressive function (By similarity). Also acts as a negative regulator of plasmacytoid dendritic cell (pDCs) activation (By similarity) The LAG3-mediated inhibitory pathway is exploited by tumors to attenuate anti-tumor immunity and escape destruction by the immune system, thereby facilitating tumor survival (PubMed:35981087, PubMed:40101708). The blockage of the LAG3- and PDCD1-mediated pathways results in the reversal of the exhausted T-cell phenotype and the normalization of the anti-tumor response, providing a rationale for cancer immunotherapy (PubMed:35981087, PubMed:40101708) May function as a ligand for MHC class II (MHC-II) on antigen-presenting cells (APC), promoting APC activation/maturation and driving Th1 immune response","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P18627/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LAG3","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LAG3","total_profiled":1310},"omim":[{"mim_id":"605776","title":"FIBRINOGEN-LIKE 1; FGL1","url":"https://www.omim.org/entry/605776"},{"mim_id":"163890","title":"SYNUCLEIN, ALPHA; SNCA","url":"https://www.omim.org/entry/163890"},{"mim_id":"153337","title":"LYMPHOCYTE ACTIVATION GENE 3; LAG3","url":"https://www.omim.org/entry/153337"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"choroid plexus","ntpm":12.9},{"tissue":"lymphoid tissue","ntpm":22.2},{"tissue":"ovary","ntpm":18.0}],"url":"https://www.proteinatlas.org/search/LAG3"},"hgnc":{"alias_symbol":["CD223"],"prev_symbol":[]},"alphafold":{"accession":"P18627","domains":[{"cath_id":"2.60.40.10","chopping":"32-68_98-168","consensus_level":"high","plddt":86.1908,"start":32,"end":168},{"cath_id":"2.60.40.10","chopping":"170-261","consensus_level":"high","plddt":92.5557,"start":170,"end":261},{"cath_id":"2.60.40.10","chopping":"269-350","consensus_level":"medium","plddt":88.1387,"start":269,"end":350},{"cath_id":"2.60.40.10","chopping":"352-427","consensus_level":"medium","plddt":85.5099,"start":352,"end":427}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P18627","model_url":"https://alphafold.ebi.ac.uk/files/AF-P18627-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P18627-F1-predicted_aligned_error_v6.png","plddt_mean":78.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LAG3","jax_strain_url":"https://www.jax.org/strain/search?query=LAG3"},"sequence":{"accession":"P18627","fasta_url":"https://rest.uniprot.org/uniprotkb/P18627.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P18627/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P18627"}},"corpus_meta":[{"pmid":"27192565","id":"PMC_27192565","title":"Lag-3, Tim-3, and TIGIT: Co-inhibitory Receptors with Specialized Functions in Immune Regulation.","date":"2016","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/27192565","citation_count":1722,"is_preprint":false},{"pmid":"28258692","id":"PMC_28258692","title":"LAG3 (CD223) as a cancer immunotherapy target.","date":"2017","source":"Immunological reviews","url":"https://pubmed.ncbi.nlm.nih.gov/28258692","citation_count":755,"is_preprint":false},{"pmid":"30580966","id":"PMC_30580966","title":"Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3.","date":"2018","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/30580966","citation_count":712,"is_preprint":false},{"pmid":"25534622","id":"PMC_25534622","title":"Clinical blockade of PD1 and LAG3--potential mechanisms of action.","date":"2015","source":"Nature reviews. 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immunotherapies.","date":"2023","source":"Oncoimmunology","url":"https://pubmed.ncbi.nlm.nih.gov/37808404","citation_count":27,"is_preprint":false},{"pmid":"37132280","id":"PMC_37132280","title":"LAG-3 transcriptomic expression patterns across malignancies: Implications for precision immunotherapeutics.","date":"2023","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37132280","citation_count":26,"is_preprint":false},{"pmid":"30279468","id":"PMC_30279468","title":"Lymphocyte activation gene 3 (Lag3) expression is increased in prion infections but does not modify disease progression.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30279468","citation_count":26,"is_preprint":false},{"pmid":"35494015","id":"PMC_35494015","title":"LAG3-PD-1 Combo Overcome the Disadvantage of Drug Resistance.","date":"2022","source":"Frontiers in 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engineered T cells.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38510235","citation_count":20,"is_preprint":false},{"pmid":"36319064","id":"PMC_36319064","title":"Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.","date":"2022","source":"Journal for immunotherapy of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/36319064","citation_count":19,"is_preprint":false},{"pmid":"39187595","id":"PMC_39187595","title":"Clinical response and pathway-specific correlates following TIGIT-LAG3 blockade in myeloma: the MyCheckpoint randomized clinical trial.","date":"2024","source":"Nature cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39187595","citation_count":18,"is_preprint":false},{"pmid":"31053880","id":"PMC_31053880","title":"PD-L1, LAG3, and HLA-DR are increasingly expressed during smoldering myeloma progression.","date":"2019","source":"Annals of 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Dimerization Is Required for TCR/CD3 Interaction and Inhibition of Antitumor Immunity.","date":"2024","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/38775415","citation_count":16,"is_preprint":false},{"pmid":"38997291","id":"PMC_38997291","title":"Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38997291","citation_count":16,"is_preprint":false},{"pmid":"38026700","id":"PMC_38026700","title":"LAG-3 expression in microglia regulated by IFN-γ/STAT1 pathway and metalloproteases.","date":"2023","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/38026700","citation_count":15,"is_preprint":false},{"pmid":"32077528","id":"PMC_32077528","title":"Ectopic expression of LAG-3 in non-small-cell lung cancer cells and its clinical 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A phylogenetically conserved acidic tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowers pH at the immune synapse and causes dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, resulting in loss of co-receptor-TCR signaling and limited T cell activation.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging, pH-sensing reporters, mutational analysis of cytoplasmic domain, functional T cell activation assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (Co-IP, live imaging, mutagenesis, functional assays) in a single rigorous study establishing a defined molecular mechanism\",\n      \"pmids\": [\"35437325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LAG3 undergoes robust non-K48-linked polyubiquitination upon engagement of MHC class II or membrane-bound (but not soluble) FGL1. This ubiquitination, mediated redundantly by E3 ligases c-Cbl and Cbl-b, disrupts membrane binding of the juxtamembrane basic residue-rich sequence, stabilizing the LAG3 cytoplasmic tail in a membrane-dissociated conformation that enables inhibitory signaling. LAG3 ubiquitination is required for suppression of antitumor immunity in vivo, and therapeutic antibodies repress LAG3 ubiquitination.\",\n      \"method\": \"Ubiquitination assays, E3 ligase identification (c-Cbl/Cbl-b), mutagenesis of juxtamembrane domain, in vivo tumor models, patient cohort analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reconstitution-level biochemistry with mutagenesis, E3 ligase identification, and in vivo validation in a single rigorous study\",\n      \"pmids\": [\"40101708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LAG3's spatial proximity to the TCR (but not CD4 co-receptor), facilitated by cognate peptide-MHC class II, is required for optimal CD4+ T cell suppression. LAG3 forms condensates with TCR signaling component CD3ε through its intracellular FSAL motif, disrupting CD3ε/Lck association. MHC class II interaction alone is insufficient for optimal LAG3 function.\",\n      \"method\": \"Proximity ligation assays, super-resolution microscopy, co-immunoprecipitation, FSAL-motif mutagenesis, bispecific antibody rescue experiments, autoimmune mouse models\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (structural proximity, Co-IP, mutagenesis, in vivo rescue) in a single rigorous study\",\n      \"pmids\": [\"40592325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LAG3 dimerization is required for its association with the TCR/CD3 complex and for optimal inhibitory function. A LAG3 mutant unable to dimerize shows perturbed TCR/CD3 association in CD8+ T cells and reduced inhibition in a tumor model. The therapeutic antibody C9B7W disrupts LAG3 dimerization and its TCR/CD3 association without blocking MHC class II binding.\",\n      \"method\": \"Dimerization-defective LAG3 mutant, co-immunoprecipitation with TCR/CD3, in vivo B16-gp100 tumor model, antibody epitope mapping\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, loss-of-function mutant, and in vivo functional validation in one study\",\n      \"pmids\": [\"38775415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal and cryo-EM structures of human and murine LAG3 ectodomains reveal a dimeric assembly mediated by Ig domain 2. A flexible 'loop 2' region in LAG3 domain 1 is the epitope for a potent antagonist antibody that blocks both MHC class II and FGL1 interactions. FGL1 cross-linking induces higher-order LAG3 oligomers, implicating ligand-mediated clustering as a mechanism for disrupting T cell activation.\",\n      \"method\": \"X-ray crystallography, cryo-EM, mutational mapping of LAG3-FGL1 and LAG3-MHC class II interfaces, oligomerization assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure and cryo-EM with mutational validation and functional interface mapping\",\n      \"pmids\": [\"35761082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent of MHC class II. FGL1 inhibits antigen-specific T cell activation, and blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity in mouse models.\",\n      \"method\": \"Binding assays, LAG-3/FGL1 blocking monoclonal antibodies, FGL1 knockout mice, in vivo tumor models, T cell activation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding, KO mice, in vivo tumor models, antibody blockade) replicated across systems\",\n      \"pmids\": [\"30580966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Binding of LAG-3 to stable peptide-MHC class II (pMHCII) complexes, but not to FGL1, induces T cell suppression in vitro. LAG-3 mutants lacking stable pMHCII-binding capacity but not FGL1-binding capacity lost suppressive activity. Targeted disruption of stable pMHCII-binding of LAG-3 in NOD mice recapitulated diabetes exacerbation seen with LAG-3 deficiency, and augmented anti-cancer immunity in C57BL/6 mice, identifying stable pMHCII as the functional ligand for both autoimmunity and anti-cancer immunity.\",\n      \"method\": \"In vitro T cell suppression assays with LAG-3 binding mutants, knock-in mice with site-specific mutations, autoimmune diabetes model (NOD), tumor models\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution-level in vitro assays with mutagenesis, corroborated by multiple in vivo genetic mouse models\",\n      \"pmids\": [\"35413245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"LAG-3 is proteolytically cleaved within the D4 transmembrane domain connecting peptide, producing a 54-kDa N-terminal extracellular fragment that oligomerizes with full-length LAG-3 (70 kDa) on the cell surface via the D1 domain, and a 16-kDa C-terminal fragment containing the transmembrane and cytoplasmic domains. The 54-kDa fragment is subsequently released as soluble LAG-3 (sLAG-3), a process enhanced by T cell activation in vitro and in vivo.\",\n      \"method\": \"Biochemical fractionation, Western blotting, domain-deletion mutants, in vivo serum analysis in C57BL/6 and RAG-1−/− mice\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical reconstitution with mutants and in vivo validation in single rigorous study\",\n      \"pmids\": [\"15557174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"LAG-3 function on T cells is mediated via its cytoplasmic domain, and a conserved 'KIEELE' motif within this domain is essential for LAG-3's regulatory activity on T cell expansion.\",\n      \"method\": \"Retroviral reconstitution of LAG-3 wild-type and cytoplasmic domain mutants in LAG-3−/− T cells, in vitro expansion assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis with retroviral reconstitution and functional readout\",\n      \"pmids\": [\"12672063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"LAG-3 negatively regulates T cell homeostasis; LAG-3-deficient mice accumulate twice as many T cells with age, and LAG-3−/− T cells show enhanced homeostatic expansion in lymphopenic hosts. Ectopic expression of wild-type LAG-3, but not a signaling-defective mutant, abrogates this deregulated expansion. Regulatory T cells depend on LAG-3 for optimal suppression of T cell homeostasis.\",\n      \"method\": \"LAG-3−/− mice, adoptive transfer into lymphopenic hosts, retroviral ectopic expression of wild-type vs. signaling-defective LAG-3, anti-LAG-3 mAb treatment in vivo\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO, adoptive transfer, and mutant reconstitution with consistent results across multiple experiments\",\n      \"pmids\": [\"15634887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"LAG-3 negatively regulates T cell expansion in vivo: LAG-3−/− mice show delayed cell cycle arrest and increased T cell expansion after superantigen stimulation, increased memory T cells after viral infection, and enlarged memory T cell pools. LAG-3 controls the size of the memory T cell pool.\",\n      \"method\": \"LAG-3−/− mice, superantigen SEB stimulation in vivo, adoptive transfer of LAG-3−/− OT-II T cells, Sendai virus and murine gammaherpesvirus infection models\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — four independent experimental systems in LAG-3−/− mice all showing consistent T cell expansion phenotype\",\n      \"pmids\": [\"15100286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"LAG-3Ig (soluble LAG-3) binds MHC class II molecules in plasma membrane lipid rafts on immature human dendritic cells (DCs) and induces DC maturation: rapid morphological changes, upregulation of costimulatory molecules, IL-12 and TNF-alpha production, and enhanced capacity to stimulate naive T cells for Th1 responses. These effects were not observed with MHC class II-specific mAbs.\",\n      \"method\": \"LAG-3Ig fusion protein treatment of human monocyte-derived DCs, flow cytometry, cytokine ELISA, T cell co-culture assays, confocal microscopy\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional readouts (morphology, surface markers, cytokines, T cell priming) in a single study\",\n      \"pmids\": [\"11937541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"LAG-3-induced MHC class II signaling in human DCs involves activation of PLCγ2, p72syk, PI3K/Akt, p42/44 ERK, and p38 MAPK pathways, all required for DC maturation. LAG-3 engagement versus anti-MHC class II antibody produces different phosphorylation patterns of c-Akt, indicating qualitative signaling differences dependent on the natural ligand.\",\n      \"method\": \"Phospho-protein analysis, kinase inhibitor studies, Western blotting in human monocyte-derived DCs, confocal microscopy with soluble LAG-3\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple pathways dissected with specific inhibitors and biochemical readouts in one study\",\n      \"pmids\": [\"12775570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"LAG3 intrinsically limits regulatory T cell (Treg) proliferation and function at inflammatory sites. Treg-specific LAG3 deletion (using Foxp3-Cre) reduces autoimmune diabetes in NOD mice by enhancing Treg proliferation, IL-2-STAT5 signaling, and Eos expression in intra-islet Tregs. LAG3-deficient Tregs outcompete wild-type Tregs specifically at the inflammatory site.\",\n      \"method\": \"Treg-specific LAG3 conditional knockout (Foxp3-Cre x Lag3-flox), RNA sequencing of intra-islet vs. peripheral Tregs, cotransfer competition experiments, STAT5 phosphorylation assays\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific KO with transcriptomic analysis and multiple functional readouts\",\n      \"pmids\": [\"28783703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"LAG-3 sustains TOX expression in exhausted CD8 T cells during chronic infection and cancer, maintaining Tex cell durability. LAG-3 controls a CD94/NKG2+ subset of exhausted T cells with enhanced cytotoxicity via recognition of the stress ligand Qa-1b. PD-1 and LAG-3 have distinct non-redundant roles: PD-1 primarily regulates proliferation while LAG-3 primarily regulates effector functions of exhausted T cells.\",\n      \"method\": \"LAG-3 and PD-1 single/double knockout mice in chronic LCMV infection, flow cytometry, TOX expression analysis, cytotoxicity assays, NKG2/Qa-1b interaction studies\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic dissection with single and double KO mice, multiple functional readouts, and human validation\",\n      \"pmids\": [\"39121847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Lag3 supports Foxp3+ regulatory T cell suppressive function by restraining Myc-dependent metabolic programming. Lag3 mutation in Tregs increases Myc expression to levels seen in Th1 effector cells, activates PI3K-Akt-Rictor signaling, and dysregulates glycolytic metabolism. Inhibiting PI3K, Rictor, or the Myc target enzyme Ldha restores normal metabolism and suppressive function in Lag3-mutant Tregs.\",\n      \"method\": \"Treg-specific Lag3 mutant mouse models, RNA sequencing, metabolic profiling, PI3K/Rictor/Ldha inhibitor rescue experiments, in vivo autoimmunity models\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Treg-specific genetic models with transcriptomics, metabolomics, and pharmacological pathway rescue\",\n      \"pmids\": [\"39236718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"LAG-3 co-localizes with CD3, CD4 or CD8 in cholesterol-rich lipid raft aggregations during primary T cell activation. Blocking LAG-3/MHC class II interactions with anti-LAG-3 mAb augments CD69 expression, T cell expansion, cell cycle entry, and Th1 (but not Th2) cytokine production in both CD4+ and CD8+ primary human T cells at low antigen concentrations.\",\n      \"method\": \"Confocal microscopy for co-localization, anti-LAG-3 blocking mAb, BrdU incorporation, flow cytometry, cytokine ELISA in primary human T cells\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — localization with functional consequence shown, single lab with multiple readouts\",\n      \"pmids\": [\"15885122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD4+CD25−LAG3+ regulatory T cells (LAG3+ Tregs) suppress humoral immune responses and B cell activity through TGF-β3 production in an Egr2- and Fas-dependent manner. This suppression requires PD-1 expression on B cells.\",\n      \"method\": \"In vitro co-culture suppression assays, TGF-β3 neutralization, Egr2-deficient mice, Fas-deficient cells, PD-1-deficient B cells, murine lupus model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mechanism shown with multiple genetic perturbations in one study, single lab\",\n      \"pmids\": [\"25695838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LAG-3 expressed on Foxp3+ regulatory T cells mediates contact-dependent suppression of CX3CR1+ intestinal macrophages via MHC class II engagement, inhibiting IL-23 and IL-1β production from macrophages and thereby restraining ILC3-mediated IL-22 production and colitis.\",\n      \"method\": \"Treg-macrophage co-culture contact-dependency assays, LAG-3 blocking antibodies, Treg-specific depletion, colitis mouse model with ILC3 transfer\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-culture and in vivo model with LAG-3 blockade showing defined suppression pathway, single lab\",\n      \"pmids\": [\"30097293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Soluble LAG-3 (sLAG-3), but not an MHC class II-specific mAb, reduces differentiation of monocytes into macrophages (in GM-CSF) and into dendritic cells (in GM-CSF + IL-4), as shown by decreased CD14 and CD1a expression. DCs differentiated in the presence of sLAG-3 have impaired antigen-presentation capacity.\",\n      \"method\": \"sLAG-3 treatment of human monocytes, flow cytometry for differentiation markers, T cell proliferation assays\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional assay with soluble protein, single lab, multiple readouts but no mutagenesis or mechanistic dissection\",\n      \"pmids\": [\"15720438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Soluble LAG-3Ig engagement of MHC class II on immature human DCs induces a distinct chemokine profile: production of IL-8 and MIP-1α/CCL3 (inflammatory), and MDC/CCL22 and TARC/CCL17 (lymph node-homing), with CCR5 downregulation and CCR7 upregulation on DC surface, potentially directing DC migration to lymph nodes.\",\n      \"method\": \"LAG-3Ig treatment of human monocyte-derived DCs, cytokine ELISA, flow cytometry for chemokine receptors, chemotaxis assays\",\n      \"journal\": \"Vaccine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional characterization with multiple chemokine readouts, single lab\",\n      \"pmids\": [\"12547595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Amyloid β precursor-like protein 1 (Aplp1) interacts with Lag3 and facilitates binding, internalization, transmission, and toxicity of pathologic α-synuclein preformed fibrils (PFF). Deletion of both Aplp1 and Lag3 eliminates dopaminergic neuron loss and behavioral deficits induced by α-syn PFF. Anti-Lag3 prevents α-syn PFF internalization by disrupting the Aplp1-Lag3 interaction.\",\n      \"method\": \"Co-immunoprecipitation of Aplp1-Lag3, double KO mice (Aplp1 and Lag3), α-synuclein PFF injection model, anti-Lag3 antibody blockade, behavioral tests, dopaminergic neuron counts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus genetic double-KO with functional readout, single lab\",\n      \"pmids\": [\"38821932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"LAG3 is not expressed by neurons in human or murine brains. Overexpression of LAG3 in human neural cells did not worsen α-synuclein pathology ex vivo. LAG3 knockout did not affect survival of A53T α-synuclein transgenic mice or seeded α-synuclein lesions in hippocampal slice cultures, though LAG3 does interact with α-synuclein fibrils with limited specificity.\",\n      \"method\": \"Immunohistochemistry, RNA-seq, Western blot, LAG3 overexpression in neural cells, LAG3 KO mice, hippocampal slice culture seeding assay, survival analysis\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple negative results with rigorous methods across different model systems consistently showing no neuronal expression or functional role\",\n      \"pmids\": [\"34309222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LAG-3 expression in microglia is induced by IFN-γ via the STAT1 pathway. Both membrane and soluble forms of LAG-3 are upregulated in IFN-γ-activated microglia. Soluble LAG-3 production is regulated by metalloproteinases ADAM10 and ADAM17. LAG-3 knockdown in microglia promotes nitric oxide production by IFN-γ, indicating LAG-3 restrains microglial activation.\",\n      \"method\": \"siRNA knockdown of STAT1, metalloproteinase inhibitors (ADAM10/17), IFN-γ stimulation of BV2 and primary microglia, in vivo intracisterna magna IFN-γ injection, nitric oxide assay\",\n      \"journal\": \"Frontiers in cellular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA knockdown and pharmacological inhibition with functional readout, single lab\",\n      \"pmids\": [\"38026700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A natural subset of plasma cells distinctively expressing LAG-3 (along with CD200, PD-L1, and PD-L2) produces IL-10 and mediates immune suppression in vivo. These LAG-3+ regulatory plasma cells develop from B cell subsets in a BCR-dependent manner and upregulate IL-10 via a TLR-driven mechanism upon challenge.\",\n      \"method\": \"Flow cytometry identification of LAG-3+ plasma cell subset, BCR-dependent development assays, TLR stimulation, IL-10 ELISA, in vivo challenge models, transcriptomic and epigenomic profiling\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — cell subset identification with functional in vivo experiments, single lab\",\n      \"pmids\": [\"30005826\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LAG3 (CD223) is an inhibitory co-receptor that localizes to the immunological synapse, where it associates with the TCR-CD3 complex (requiring LAG3 dimerization and facilitated by cognate pMHC class II proximity) and suppresses T cell activation by: (1) using an acidic glutamic acid-proline repeat in its cytoplasmic tail to lower local pH and dissociate Lck from CD4/CD8 co-receptors; (2) forming condensates with CD3ε via its FSAL motif to disrupt CD3ε/Lck association; and (3) undergoing c-Cbl/Cbl-b-mediated polyubiquitination upon ligand engagement, which releases a juxtamembrane basic sequence from the membrane to permit inhibitory signaling. Its primary functional ligand is stable peptide-MHC class II (pMHCII), with FGL1 serving as an additional ligand that induces LAG3 oligomerization; the LAG3 ectodomain forms a dimer mediated by Ig domain 2 with a flexible loop 2 region in domain 1 engaging both ligands. Beyond T cell-intrinsic signaling, LAG3 on Tregs limits their proliferation and metabolic activity (via restraint of Myc/PI3K-Akt-Rictor signaling) and mediates contact-dependent suppression of antigen-presenting cells through MHC class II; as a soluble form generated by ADAM10/ADAM17-mediated cleavage, it can also induce DC maturation through MHC class II signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LAG3 (CD223) is an inhibitory co-receptor that restrains T cell activation, controls T cell homeostasis and the size of the memory pool, and supports the suppressive function of regulatory T cells [#0, #10, #13]. Upon recruitment to the immunological synapse, LAG3 associates with the TCR-CD3 complex independently of MHC class II, a step requiring LAG3 dimerization, and delivers inhibitory signals through its cytoplasmic tail [#0, #3]. Mechanistically, an acidic tandem glutamic acid-proline repeat lowers local synapse pH to dissociate Lck from the CD4/CD8 co-receptor, while LAG3 also forms condensates with CD3\\u03b5 via an intracellular FSAL motif to disrupt CD3\\u03b5/Lck association, with cognate peptide-MHC class II proximity to the TCR being required for optimal suppression [#0, #2]. Ligand engagement triggers c-Cbl/Cbl-b-mediated non-K48 polyubiquitination that releases the membrane-bound juxtamembrane basic sequence, stabilizing the tail in a signaling-competent conformation; this ubiquitination is required for suppression of antitumor immunity [#1]. Stable peptide-MHC class II is the functional ligand for both autoimmunity and anti-cancer immunity, with FGL1 serving as an MHC-class-II-independent ligand whose cross-linking drives higher-order LAG3 oligomerization; the ectodomain dimerizes through Ig domain 2, with a domain-1 loop 2 region engaging both ligands [#4, #5, #6]. On Foxp3+ Tregs, LAG3 limits proliferation and metabolic activity by restraining Myc and PI3K-Akt-Rictor signaling and mediates contact-dependent suppression of antigen-presenting cells via MHC class II [#13, #15, #18]. LAG3 is proteolytically cleaved by ADAM10/ADAM17 to release a soluble ectodomain that engages MHC class II on dendritic cells to drive their maturation and signaling [#7, #11, #23].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that soluble LAG-3 acts as an MHC class II ligand on dendritic cells, defining a function for LAG3 beyond T-cell-intrinsic inhibition.\",\n      \"evidence\": \"LAG-3Ig fusion protein treatment of human monocyte-derived DCs with morphology, surface marker, cytokine, and T cell priming readouts\",\n      \"pmids\": [\"11937541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the LAG3 cytoplasmic signaling mechanism\", \"Effects shown with soluble fusion protein rather than endogenous cleaved sLAG-3\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified the cytoplasmic determinant of LAG3 inhibitory signaling, showing a conserved KIEELE motif is essential for regulating T cell expansion.\",\n      \"evidence\": \"Retroviral reconstitution of wild-type and cytoplasmic mutants in LAG-3\\u2212/\\u2212 T cells with expansion assays; parallel DC studies dissecting MHC-class-II signaling pathways\",\n      \"pmids\": [\"12672063\", \"12775570\", \"12547595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effectors of the KIEELE motif not identified at the time\", \"Did not connect motif to TCR-CD3 association\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed LAG-3 negatively regulates T cell expansion and memory pool size in vivo, and that it is proteolytically processed to release a soluble ectodomain.\",\n      \"evidence\": \"LAG-3\\u2212/\\u2212 mice with superantigen and viral infection models; biochemical fractionation and domain-deletion mapping of cleavage fragments\",\n      \"pmids\": [\"15100286\", \"15557174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Protease responsible for cleavage not identified in these studies\", \"Molecular mechanism of inhibition still unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked LAG3 to homeostatic T cell control and lipid-raft synapse localization, and extended sLAG-3 effects to monocyte differentiation.\",\n      \"evidence\": \"LAG-3\\u2212/\\u2212 mice with adoptive transfer; confocal co-localization with CD3/CD4/CD8 in lipid rafts; sLAG-3 treatment of human monocytes\",\n      \"pmids\": [\"15634887\", \"15885122\", \"15720438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Co-localization data (Medium) not mechanistically dissected\", \"Treg dependence on LAG3 shown functionally but molecular basis unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined a Treg-intrinsic role: LAG3 limits Treg proliferation and function at inflammatory sites by restraining IL-2-STAT5 signaling.\",\n      \"evidence\": \"Treg-specific conditional knockout (Foxp3-Cre x Lag3-flox), RNA-seq of intra-islet Tregs, competition transfers, STAT5 phosphorylation assays\",\n      \"pmids\": [\"28783703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How LAG3 signaling links to STAT5 restraint not resolved\", \"Site-specific competitive advantage mechanism unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified FGL1 as a major MHC-class-II-independent functional ligand and established contact-dependent Treg suppression of APCs through MHC class II.\",\n      \"evidence\": \"FGL1 binding assays, blocking antibodies, FGL1 KO mice, tumor models; Treg-macrophage co-culture with LAG-3 blockade and colitis model\",\n      \"pmids\": [\"30580966\", \"30097293\", \"30005826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of FGL1 versus pMHCII not yet ranked\", \"Receptor conformational consequences of ligand binding unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the structural and signaling framework: ectodomain dimerization via Ig domain 2, ligand-induced oligomerization, TCR-CD3 association requiring dimerization, and a pH/Lck-dissociation mechanism in the cytoplasmic tail.\",\n      \"evidence\": \"Crystallography and cryo-EM with interface mutagenesis; dimerization-defective mutants with reciprocal Co-IP and tumor models; Co-IP, live imaging, pH reporters and functional T cell assays\",\n      \"pmids\": [\"35761082\", \"38775415\", \"35437325\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How dimerization and the acidic E-P repeat are coordinated in time not fully resolved\", \"Stoichiometry of LAG3-TCR-CD3 assembly at the synapse not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved which ligand drives suppression, showing stable pMHCII binding is the functional ligand for both autoimmunity and anti-cancer immunity.\",\n      \"evidence\": \"In vitro suppression assays with binding mutants and knock-in mice in NOD diabetes and tumor models\",\n      \"pmids\": [\"35413245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with FGL1 ligand activity not fully integrated\", \"Whether different ligands trigger distinct signaling outputs unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Distinguished LAG3 from other checkpoints in exhausted T cells and detailed Treg metabolic control by LAG3.\",\n      \"evidence\": \"LAG-3/PD-1 single and double KO mice in chronic LCMV with TOX and cytotoxicity readouts; Treg-specific Lag3 mutants with transcriptomics, metabolomics, and PI3K/Rictor/Ldha inhibitor rescue\",\n      \"pmids\": [\"39121847\", \"39236718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single inhibitory receptor selectively shapes effector function versus proliferation not mechanistically defined\", \"Link between membrane signaling and Myc/metabolic restraint not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Completed the proximal signaling model: ligand-induced c-Cbl/Cbl-b ubiquitination releases the juxtamembrane tail for signaling, and CD3\\u03b5 condensate formation via FSAL disrupts CD3\\u03b5/Lck.\",\n      \"evidence\": \"Ubiquitination assays, E3 ligase identification, juxtamembrane mutagenesis, in vivo tumor models and patient cohorts; proximity ligation, super-resolution imaging, FSAL mutagenesis, bispecific antibody rescue, autoimmune models\",\n      \"pmids\": [\"40101708\", \"40592325\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of ubiquitination, pH-Lck dissociation, and CD3\\u03b5 condensation into one ordered pathway not yet formalized\", \"Generality across CD4 versus CD8 T cells partially addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Explored a non-immune role in neurodegeneration, with conflicting evidence on LAG3 as a mediator of pathologic \\u03b1-synuclein uptake.\",\n      \"evidence\": \"Aplp1-Lag3 Co-IP, double-KO mice and anti-Lag3 blockade in \\u03b1-syn PFF model; contrasted with prior reports of no neuronal LAG3 expression or functional effect, and IFN-\\u03b3/STAT1-induced microglial LAG3\",\n      \"pmids\": [\"38821932\", \"34309222\", \"38026700\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conflicting reports on whether LAG3 contributes to \\u03b1-synuclein pathology not reconciled\", \"Cell type expressing functionally relevant LAG3 in brain disputed\", \"Mechanism of Aplp1-Lag3 cooperation in fibril internalization incomplete\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the distinct proximal mechanisms (acidic E-P repeat-driven Lck dissociation, FSAL-CD3\\u03b5 condensation, Cbl-mediated ubiquitination) are temporally ordered and whether they operate in parallel or sequentially during a single inhibitory event remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified kinetic model linking the three cytoplasmic mechanisms\", \"Relative ligand-specific contributions of pMHCII versus FGL1 to each step not quantified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 8, 9]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [6, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 6, 10, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CD3E\", \"FGL1\", \"CBL\", \"CBLB\", \"APLP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}