{"gene":"CD226","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":1996,"finding":"DNAM-1 (CD226) is a 65 kDa transmembrane glycoprotein with two immunoglobulin-like domains that is tyrosine-phosphorylated upon activation and functions as a signal-transducing adhesion molecule participating in primary adhesion during CTL-mediated cytotoxicity. Anti-DNAM-1 mAb inhibits T and NK cell-mediated cytotoxicity and blocks cytokine production by alloantigen-specific T cells.","method":"Monoclonal antibody blocking, immunoprecipitation, signal transduction analysis","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — original characterization with multiple orthogonal methods (mAb inhibition, phosphorylation assay, cytotoxicity assay), replicated across subsequent studies","pmids":["8673704"],"is_preprint":false},{"year":1985,"finding":"TLiSA1 (CD226) is a T lineage-specific activation antigen whose antibody-mediated blockade from the beginning of mixed lymphocyte culture inhibits the induction of both CTL and anomalous killer (AK) cells from precursors, reducing CTL frequency and greatly reducing AK cell frequency under limiting dilution conditions, indicating a role in differentiation of cytotoxic effectors.","method":"Mixed lymphocyte culture with mAb blockade, limiting dilution analysis","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional blockade with mAb and F(ab')2 fragments, limiting dilution confirmation, single lab","pmids":["2580933"],"is_preprint":false},{"year":1989,"finding":"TGF-β suppresses TLiSA1 (CD226) surface expression and inhibits CTL and LAK cell differentiation in a dose-dependent manner; antibody to TGF-β reverses both effects, and anti-TLiSA1 antibody mimics TGF-β in inhibiting cytotoxic effector differentiation, linking TLiSA1 expression to the differentiation process.","method":"Mixed lymphocyte culture with TGF-β treatment, mAb blocking, anti-TGF-β rescue","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cytokine and antibody perturbation with rescue experiment, single lab","pmids":["2541074"],"is_preprint":false},{"year":1997,"finding":"TLiSA1/PTA1 (CD226) was cloned and identified as a novel immunoglobulin superfamily member with two V-set domains only. Identity between the T cell activation antigen TLiSA1 and platelet antigen PTA1 was established by immunological criteria, internal peptide sequencing, and RT-PCR of platelet transcript. Phorbol ester stimulates mRNA and surface expression, while combined phorbol ester and ionophore suppresses expression via a calcineurin-independent pathway.","method":"cDNA cloning, peptide sequencing, RT-PCR, flow cytometry, pharmacological perturbation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — cDNA cloning with structural determination, peptide sequence confirmation, multiple orthogonal methods","pmids":["9268302"],"is_preprint":false},{"year":1999,"finding":"LFA-1 physically associates with DNAM-1 in NK cells and anti-CD3-stimulated T cells, and serine phosphorylation of DNAM-1 plays a critical role in this association. Cross-linking of LFA-1 induces tyrosine phosphorylation of DNAM-1, for which the Fyn protein tyrosine kinase is responsible. DNAM-1-mediated cytotoxicity is defective in LFA-1-deficient (LAD syndrome) NK cells and is restored by genetic reconstitution of LFA-1 expression.","method":"Co-immunoprecipitation, genetic reconstitution, phosphorylation assays, cytotoxicity assays with LAD patient NK cells","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reciprocal co-IP, phosphorylation mapping, genetic rescue experiment, multiple orthogonal approaches","pmids":["10591186"],"is_preprint":false},{"year":2003,"finding":"CD226 mediates platelet adhesion to vascular endothelial cells; thrombin-activated (but not resting) platelets bind to intact HUVEC via CD226, and thrombin-induced platelet activation induces tyrosine phosphorylation of CD226. Mutant transfectant experiments demonstrated that tyrosine at residue 322 is critical for CD226 adhesive function. CD226 is also expressed on megakaryocytic cells and mediates their adhesion to HUVEC.","method":"mAb blocking adhesion assay, site-directed mutagenesis (Y322 mutant transfectants), phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis of specific residue combined with functional adhesion assay, phosphorylation measurement, multiple cell types","pmids":["12847109"],"is_preprint":false},{"year":2003,"finding":"Upon TCR antigen recognition, LFA-1 physically associates with CD226 (DNAM-1) and the protein tyrosine kinase Fyn. Lentiviral expression of mutant Y322F CD226 in naive CD4+ and CD8+ T cells inhibited LFA-1-mediated costimulatory signals for naive T cell proliferation and IL-12-independent Th1 differentiation. LFA-1, CD226, and Fyn are polarized at the immunological synapse upon CD3 stimulation. Lipid raft disruption inhibits LFA-1 costimulatory signaling in CD4+ but not CD8+ T cells.","method":"Lentiviral mutant transduction (Y322F), T cell proliferation assay, Th1 differentiation assay, immunological synapse imaging, lipid raft disruption","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — site-directed mutagenesis with lentiviral rescue, multiple functional readouts, co-IP of complex, imaging","pmids":["14676297"],"is_preprint":false},{"year":2004,"finding":"Human CD155 (PVR) and CD112 (Nectin-2/PRR-2) were identified as ligands for human CD226. Ectopic expression of CD155 and/or CD112 on mouse BW5147 T cells increased their susceptibility to IL-2-activated T and NK cell-mediated cytotoxicity, which was specifically inhibited by anti-CD226 mAb. Ligation of CD226 and LFA-1 cooperates in triggering cytotoxicity and cytokine secretion.","method":"Ectopic expression/transfection, cytotoxicity assay, mAb blocking, binding affinity measurement","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reconstitution via transfection, specific inhibition by anti-CD226 mAb, replicated across multiple labs","pmids":["15039383"],"is_preprint":false},{"year":2004,"finding":"DNAM-1 (CD226) regulates monocyte transendothelial migration; PVR (CD155) is expressed at endothelial cell junctions and is the major DNAM-1 ligand on endothelial cells (Nectin-2 contributes less). Anti-DNAM-1 and anti-PVR mAbs blocked monocyte transmigration in vitro, arresting monocytes at the apical surface over intercellular junctions, indicating DNAM-1-PVR interaction occurs during the diapedesis step.","method":"Soluble DNAM-1-Fc binding assay, mAb blocking, transendothelial migration assay, confocal microscopy","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — soluble receptor binding with specific antibody competition, functional transmigration blockade with stage-specific phenotype, multiple orthogonal approaches","pmids":["15136589"],"is_preprint":false},{"year":2005,"finding":"PVR and Nectin-2 were confirmed as DNAM-1 ligands by mass spectrometry identification of purified proteins and by demonstrating mutual staining between PVR-Fc/Nectin-2-Fc chimeric molecules and DNAM-1-expressing cells and vice versa. DNAM-1/PVR or Nectin-2 interaction enhanced NK-mediated lysis of tumor cells.","method":"Tryptic digestion and mass spectrometry, chimeric protein staining, cytotoxicity assay with mAb blocking","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mass spectrometry identification, reciprocal binding validation, functional cytotoxicity assay, replicated","pmids":["15607800"],"is_preprint":false},{"year":2005,"finding":"DNAM-1 ligand-transduced RMA tumor cells were rejected in syngeneic mice in a CD8+ T cell- and NK cell-dependent manner generating CD8+ memory CTL. DNAM-1 is expressed on CD8α+ dendritic cells, and cross-linking DNAM-1 on these DCs induced their maturation and Th1 cell priming. Tumor rejection was abrogated in CD4+ T cell-depleted and MHC class II-deficient mice.","method":"Ligand transduction, in vivo tumor rejection assay, DC maturation assay, T cell depletion, KO mouse model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple in vivo genetic and depletion approaches, DC functional assay, multiple cell type contributions defined","pmids":["16249389"],"is_preprint":false},{"year":2005,"finding":"Serine phosphorylation at residue 329 (S329) of DNAM-1 is required for LFA-1-dependent lipid raft recruitment of DNAM-1. Wild-type but not S329 mutant DNAM-1 associates with lipid rafts at peripheral supra-molecular activation clusters, and lipid raft association is necessary for tyrosine phosphorylation of DNAM-1, which is essential for LFA-1-mediated costimulatory signaling for naive T cell proliferation and differentiation.","method":"Site-directed mutagenesis (S329), lipid raft fractionation, immunological synapse imaging, T cell functional assays","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — specific residue mutagenesis, lipid raft biochemical fractionation, functional consequence demonstrated","pmids":["15684041"],"is_preprint":false},{"year":2005,"finding":"Murine DNAM-1 and its PVR homologue were identified; they physically bind each other and ligand binding of murine DNAM-1 induces a costimulatory signal in antigen-specific CD8+ T cells.","method":"Binding assays, T cell costimulation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed, functional costimulation assay, single lab","pmids":["15752754"],"is_preprint":false},{"year":2006,"finding":"Human mast cells and eosinophils express DNAM-1 (CD226) and its ligand Nectin-2 (CD112), respectively. CD226 engagement on mast cells synergizes with FcεRI and augments degranulation through a pathway involving Fyn, LAT, PLCγ2, and CD18. Blocking CD112 on eosinophils normalized the hyperactivity of IgE-activated mast cells co-cultured with eosinophils. This pathway is completely inhibited by linking IgE with IRp60 using a bispecific antibody.","method":"mAb blocking, co-culture assay, pharmacological inhibition of signaling intermediates, bispecific antibody","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined signaling pathway with pharmacological inhibitors, co-culture system, single lab","pmids":["16831868"],"is_preprint":false},{"year":2006,"finding":"Lipid raft recruitment of DNAM-1 in CD4+ T cells depends on LFA-1 expression; in LFA-1-deficient CD4+ T cells, TCR-mediated serine phosphorylation of DNAM-1 occurs but DNAM-1 does not associate with lipid rafts, indicating serine phosphorylation of DNAM-1 primarily induces physical association with LFA-1, which then carries DNAM-1 into lipid rafts.","method":"LFA-1-deficient mouse T cells, lipid raft fractionation, phosphorylation assay","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genetic knockout of LFA-1 combined with biochemical fractionation, mechanistic ordering of events established","pmids":["16636013"],"is_preprint":false},{"year":2008,"finding":"DNAM-1-deficient mice show accelerated tumor development and mortality after transplantation of DNAM-1 ligand (CD155)-expressing Meth A fibrosarcoma cells. DNAM-1-deficient CTL and NK cells show significantly reduced cytotoxic activity against DNAM-1 ligand-expressing tumor cells in vitro. DNAM-1-deficient mice also develop significantly more fibrosarcoma and papilloma in response to chemical carcinogens MCA and DMBA, demonstrating an in vivo tumor immunosurveillance role.","method":"DNAM-1 knockout mice, tumor transplantation model, chemical carcinogen model, in vitro cytotoxicity assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — germline KO with multiple tumor models (transplant and carcinogen-induced), replicated across labs","pmids":["19029379"],"is_preprint":false},{"year":2008,"finding":"CD8+ T cells require DNAM-1 for co-stimulation when antigen is presented by nonprofessional APCs but not dendritic cells. NK cells require DNAM-1 for elimination of tumor cells with paucity of other NK-activating ligands. DNAM-1-deficient mice generated from knockout model demonstrated these cell-type and context-specific requirements.","method":"DNAM-1 knockout mice, in vitro T cell co-stimulation assay, in vitro NK cytotoxicity assay, DC vs. non-professional APC comparison","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined cellular phenotype, independent lab from PMID 19029379, multiple functional readouts","pmids":["19029380"],"is_preprint":false},{"year":2012,"finding":"TIGIT exerts immunosuppressive effects on CD4+ T cells by competing with CD226 for binding to CD155. Knockdown of TIGIT by shRNA increased T-bet and IFN-γ expression; this increase was overcome by blocking CD226 signaling, demonstrating that TIGIT inhibits T cell function partly by displacing CD226 from its CD155 ligand. TIGIT also directly inhibits T cells in a T cell-intrinsic manner.","method":"shRNA knockdown of TIGIT, anti-TIGIT agonist mAb, anti-CD226 blocking mAb, cytokine/transcription factor measurement","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockdown combined with receptor blockade epistasis, multiple orthogonal methods, functional rescue","pmids":["22427644"],"is_preprint":false},{"year":2015,"finding":"DNAM-1 controls NK cell-mediated cytotoxicity and cytokine production via a conserved tyrosine- and asparagine-based ITT-like motif in its cytoplasmic domain. Upon Src kinase-mediated phosphorylation of this motif, DNAM-1 binds adaptor Grb2, leading to activation of Vav-1, PI3K, and PLCγ1, as well as Erk and Akt kinases and calcium flux. DNAM-1 signaling also enhances cytotoxicity by increasing actin polymerization and granule polarization. DNAM-1-mediated adhesion is signal-independent and insufficient alone for cytotoxicity.","method":"Site-directed mutagenesis of ITT-like motif, co-immunoprecipitation of Grb2, downstream signaling assays (Erk, Akt, calcium flux), actin polymerization assay, granule polarization assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis of specific signaling motif, co-IP of downstream adaptor Grb2, multiple downstream effectors characterized with orthogonal methods","pmids":["26552706"],"is_preprint":false},{"year":2015,"finding":"DNAM-1 expression identifies two functionally distinct NK cell subsets: DNAM-1+ NK cells produce high levels of inflammatory cytokines, have enhanced IL-15 signaling, and proliferate vigorously; DNAM-1- NK cells differentiate from DNAM-1+ cells, express more NK receptor-related genes, and produce higher levels of MIP1 chemokines.","method":"Transcriptional profiling, functional cytokine production assay, proliferation assay, IL-15 signaling assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptional profiling combined with functional studies, single lab","pmids":["25818301"],"is_preprint":false},{"year":2015,"finding":"In educated NK cells, upon target cell recognition, the conformational state of LFA-1 changes and both active LFA-1 and DNAM-1 rapidly colocalize at the immune synapse. DNAM-1 expression correlates with the quantity and quality of inhibitory input by HLA class I-specific KIRs and CD94/NKG2A, and with the magnitude of functional responses.","method":"Flow cytometry, live imaging, functional cytotoxicity correlation analysis","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct imaging of immune synapse localization with functional correlation, single lab","pmids":["25825444"],"is_preprint":false},{"year":2015,"finding":"CD226 is required for immunosurveillance and therapy of multiple myeloma; CD226 limits spontaneous MM development in Vk*MYC transgenic mice. The anti-myeloma immune response is mediated by both NK and CD8+ T cells through perforin and IFN-γ pathways. CD226 expression is also required for optimal antimyeloma efficacy of cyclophosphamide and bortezomib.","method":"Cd226 mutant mice crossed with Vk*MYC MM model, NK/T cell depletion, perforin/IFN-γ blockade, drug treatment","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model with long-term surveillance study, mechanistic pathway defined by depletion/blockade, drug efficacy dependence","pmids":["25893601"],"is_preprint":false},{"year":2018,"finding":"CD226 binding to CD155 is mediated by a unique side-by-side arrangement of two tandem IgV domains (D1 and D2). Crystal structure of mCD226-ecto revealed that D1 forms the conserved binding interface with CD155-D1, while D2 provides structural support and forms direct interactions with CD155. Deletion of D2 substantially reduced CD226 binding efficacy to CD155.","method":"Crystal structure determination (X-ray crystallography), deletion mutant binding assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by domain deletion, structural basis for binding established","pmids":["30591568"],"is_preprint":false},{"year":2018,"finding":"CD226 regulates NK cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1. Engagement of CD155 ligand results in phosphorylation of FOXO1 downstream of CD226. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses.","method":"CD226-deficient mice, gene expression analysis, in vitro cytotoxicity/stimulation assay, FOXO1 phosphorylation assay, pharmacological inhibitors","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genetic KO combined with pharmacological inhibition and phosphorylation assays, multiple orthogonal approaches defining a specific transcription factor as downstream effector","pmids":["30504141"],"is_preprint":false},{"year":2018,"finding":"CD226 opposes TIGIT to disrupt Treg suppressive function in melanoma; TIGIT+ Tregs are highly suppressive and stable while CD226 disrupts Treg suppression and stability. A high TIGIT/CD226 ratio in Tregs correlates with increased Treg frequencies in tumors.","method":"Flow cytometry of Tregs from melanoma patients and tumors, functional suppression assay, mAb blocking of TIGIT and CD226","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor blocking functional assay in patient samples, single lab, correlation plus functional intervention","pmids":["30046006"],"is_preprint":false},{"year":2019,"finding":"DNAM-1 on small peritoneal macrophages (SPMs) is required for CD4+ T cell co-stimulation; SPMs but not large peritoneal macrophages (LPMs) express DNAM-1 and have antigen-presenting capacity. DNAM-1 deficiency on SPMs or blockade of DNAM-1/ligand interaction impaired CD4+ T cell priming. Wild-type but not DNAM-1-deficient SPMs enhanced T and B cell responses in vivo upon adoptive transfer.","method":"DNAM-1 KO mice, co-culture antigen presentation assay, adoptive SPM transfer, in vivo immunization","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO combined with adoptive transfer, multiple in vitro and in vivo functional readouts","pmids":["30315271"],"is_preprint":false},{"year":2020,"finding":"Tumor-derived CD155 initiates phosphorylation of CD226 at tyrosine 319 (Y319) by Src kinases, which enables ubiquitination of CD226 by the E3 ligase CBL-B, followed by internalization and proteasomal degradation of CD226. Mutation of Y319 led to increased CD226 surface expression, enhanced anti-tumor immunity, and improved immune checkpoint blockade efficacy.","method":"Y319 mutagenesis, phosphorylation assay, CBL-B co-immunoprecipitation, proteasomal inhibition, in vivo tumor models","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — specific residue mutagenesis, identification of E3 ligase CBL-B, proteasomal degradation pathway validated, in vivo functional consequence","pmids":["33053330"],"is_preprint":false},{"year":2020,"finding":"Loss of CD226 expression on CD8+ T cells is driven by the transcriptional regulator Eomesodermin (Eomes) through an antigen-specific mechanism. CD226-negative CD8+ T cells exhibit reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program. CD226-deficient CD8+ TILs fail to respond to anti-PD-1, and immune checkpoint blockade efficacy was hampered in Cd226-/- mice.","method":"Transcriptome analysis, Eomes overexpression/knockdown, Cd226-/- mice, anti-PD-1 treatment, LFA-1 activation assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined transcriptional mechanism, multiple functional readouts, published alongside companion paper","pmids":["33053331"],"is_preprint":false},{"year":2020,"finding":"Soluble CD155 (sCD155) secreted by tumor cells inhibits DNAM-1-mediated NK cell degranulation and promotes lung colonization of B16/BL6 melanoma; in vivo the effect required DNAM-1 (not TIGIT or CD96). DNAM-1 has greater affinity than TIGIT and CD96 for sCD155, indicating preferential competitive binding.","method":"sCD155-expressing tumor injection in KO mice (DNAM-1, TIGIT, CD96), in vitro degranulation assay, binding affinity measurement","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple receptor-specific KO mice, in vitro and in vivo complementary approaches, binding affinity data","pmids":["32040157"],"is_preprint":false},{"year":2020,"finding":"Anti-TIGIT treatment selectively promotes CD226 phosphorylation at tyrosine 322 in CD226hiCD8+ T cells, and CD226 agonist antibody augments the effect of TIGIT blockade on CD8+ T cell responses. CD226loCD8+ T cells accumulate at the tumor site with exhausted phenotype.","method":"Flow cytometry, phospho-Y322 analysis after anti-TIGIT treatment, CD226 agonist antibody functional assay","journal":"Cancer immunology research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific phosphorylation site measured in response to TIGIT blockade, functional agonist assay, single lab","pmids":["32265229"],"is_preprint":false},{"year":2020,"finding":"CD226 deletion in mice improves post-myocardial infarction healing by favoring macrophage M2 polarization and suppressing M1 polarization. In vitro, CD226 deletion in bone marrow-derived macrophages potentiated M2 and suppressed M1 polarization. CD226 deletion also restrained inflammatory monocyte mobilization from spleen to blood.","method":"CD226 KO mice with coronary ligation model, bone marrow-derived macrophage polarization assay, flow cytometry of monocyte subsets","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with in vivo and in vitro macrophage polarization assays, single lab","pmids":["32104514"],"is_preprint":false},{"year":2021,"finding":"DNAM-1 competes with TIGIT in binding to their common ligand CD155, thereby regulating TIGIT signaling to down-regulate Treg cell function without requiring DNAM-1-mediated intracellular signaling. DNAM-1 deficiency augments TIGIT signaling, which inhibits Akt-mTORC1 pathway activation, maintaining Foxp3 expression and Treg function under inflammatory conditions. Transfer of DNAM-1-deficient Treg cells ameliorated graft-versus-host disease better than wild-type Tregs.","method":"DNAM-1-deficient Treg transfer, GvHD model, Akt-mTORC1 signaling assay, Foxp3 stability assay, TIGIT signaling measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO Treg transfer with GvHD functional readout, defined signaling pathway (Akt-mTORC1/Foxp3), mechanistic ordering established","pmids":["34011606"],"is_preprint":false},{"year":2021,"finding":"CD226 deficiency on adipose tissue macrophages (ATMs) decreases phosphorylation of VAV1, AKT, and FOXO1 and upregulates PPAR-γ, thereby inhibiting macrophage M1 polarization. Administration of a PPAR-γ inhibitor restored M1 phenotype in CD226KO ATMs, placing PPAR-γ downstream of CD226 signaling in macrophage polarization.","method":"CD226 KO mice, HFD obesity model, phosphorylation assays (VAV1, AKT, FOXO1), PPAR-γ inhibitor rescue experiment","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with pharmacological rescue defining PPAR-γ as downstream effector, single lab","pmids":["34823548"],"is_preprint":false},{"year":2022,"finding":"CD226 signals promote the early phase of human Tfh cell differentiation; blocking CD226 potently inhibited proliferation and Tfh marker expression of naive CD4+ T cells and Tfh precursors stimulated with CD155-expressing APCs. In contrast, mature GC-Tfh cells require only weak CD226 signals, and attenuating CD226 rather increased CXCR5, ICOS, and IL-21 expression, indicating the requirement for CD226 wanes as Tfh cells mature.","method":"CD155-expressing artificial APC co-culture, anti-CD226 blocking antibody, proliferation assay, Tfh marker expression assay","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — blocking antibody functional assay with defined differentiation stages, single lab","pmids":["35273617"],"is_preprint":false},{"year":2023,"finding":"Treg-specific Cd226 deletion (TregΔCd226 mice) decreased insulitis and diabetes incidence in NOD mice. CD226-deficient pancreatic Tregs had increased TIGIT expression. NOD splenocytes treated with TIGIT-Fc fusion protein showed reduced T cell proliferation and IFN-γ production, indicating CD226/TIGIT imbalance on Tregs contributes to Treg instability.","method":"Conditional Treg-specific KO (Foxp3-Cre), diabetes incidence monitoring, TIGIT-Fc fusion protein treatment, proliferation and cytokine assays","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional KO with disease endpoint, TIGIT-Fc functional validation, multiple methods","pmids":["37625150"],"is_preprint":false},{"year":2024,"finding":"CD226 is inducibly expressed in activated ILC2s and enhances their cytokine secretion and effector functions. Mechanistically, CD226 alters intracellular metabolism and enhances PI3K/AKT and MAPK signaling pathways in ILC2s. Blocking CD226 ameliorates ILC2-dependent airway hyperreactivity in IL-33 and Alternaria alternata-induced models.","method":"Anti-CD226 antibody treatment, RNA sequencing of sorted ILC2s, PI3K/AKT and MAPK signaling assay, in vivo AHR mouse models (WT and Rag2-/- mice)","journal":"The Journal of allergy and clinical immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNA sequencing, defined signaling pathway, in vivo functional assay in multiple models, multiple orthogonal approaches","pmids":["38244725"],"is_preprint":false},{"year":2014,"finding":"The alphaherpesvirus gD glycoprotein causes degradation and down-regulation of CD112 (a DNAM-1 ligand), reducing DNAM-1 binding to infected cells and suppressing DNAM-1-mediated NK cell degranulation and lysis of PRV- or HSV-2-infected cells.","method":"Viral infection/transfection with gD expression, flow cytometry of CD112, NK degranulation assay, cytotoxicity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined viral protein responsible, CD112 degradation linked to impaired DNAM-1 binding and NK function, single lab","pmids":["25352670"],"is_preprint":false},{"year":2016,"finding":"Mouse CMV m20.1 protein retains PVR (DNAM-1 ligand) in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking m20.1 was attenuated in normal but not DNAM-1 KO mice. This attenuation was partially reversed by NK cell depletion and abolished by simultaneous depletion of mononuclear phagocytes, indicating that inflammatory monocytes together with NK cells control CMV through the DNAM-1-PVR pathway.","method":"MCMV m20.1 deletion mutant, DNAM-1 KO mice, cell depletion, iNOS inhibition, CCL2 blocking","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — viral KO mutant combined with host KO mice and cell depletion, specific viral immune evasion mechanism identified","pmids":["27503073"],"is_preprint":false}],"current_model":"CD226 (DNAM-1) is a two-IgV-domain immunoglobulin superfamily transmembrane receptor that binds CD155 (PVR) and CD112 (Nectin-2) via its D1–D2 ectodomain in a side-by-side arrangement; upon ligand engagement it undergoes Src kinase-mediated phosphorylation at Y319 (enabling CBL-B-mediated ubiquitination and proteasomal degradation) and serine phosphorylation at S329 (enabling LFA-1 association and lipid raft recruitment), while signaling through a cytoplasmic ITT-like motif that recruits Grb2 and activates Vav-1, PI3K, PLCγ1, Erk, and Akt to drive actin polymerization, granule polarization, cytokine production, and FOXO1 inactivation; it competes with the inhibitory receptor TIGIT for shared ligands to promote effector lymphocyte (NK, CD8+ T, and Treg) function, and is required in vivo for tumor immunosurveillance, control of viral infections, and regulation of inflammatory responses including monocyte transendothelial migration and ILC2-dependent airway hyperreactivity."},"narrative":{"mechanistic_narrative":"CD226 (DNAM-1) is an immunoglobulin-superfamily transmembrane glycoprotein with two V-set domains that functions as an activating adhesion and signaling receptor central to cytotoxic lymphocyte function and immunosurveillance [PMID:8673704, PMID:9268302]. It engages the nectin-family ligands CD155 (PVR) and CD112 (Nectin-2) through a side-by-side tandem D1–D2 IgV arrangement, in which D1 forms the conserved interface with CD155 while D2 provides structural support and direct contacts required for high-affinity binding [PMID:15039383, PMID:30591568]. Ligand engagement triggers Src-family kinase-mediated tyrosine phosphorylation of a cytoplasmic ITT-like motif (residue Y322/Y319 critical for adhesive and signaling function) that recruits Grb2 and activates Vav-1, PI3K, PLCγ1, Erk, and Akt, driving actin polymerization, granule polarization, calcium flux, cytokine production, and phosphorylation-mediated inactivation of FOXO1 [PMID:12847109, PMID:26552706, PMID:30504141]. Serine phosphorylation at S329 instead promotes physical association with LFA-1, which carries CD226 into lipid rafts at the immunological synapse; this LFA-1 partnership is required for CD226 tyrosine phosphorylation and costimulatory signaling, and Fyn mediates the bidirectional phosphorylation between the two receptors [PMID:10591186, PMID:14676297, PMID:15684041, PMID:16636013]. CD226 competes with the inhibitory receptor TIGIT (and outcompetes CD96) for the shared CD155 ligand, and the CD226/TIGIT balance dictates effector versus regulatory outcomes in CD8+ T cells, NK cells, and Tregs [PMID:22427644, PMID:32040157, PMID:34011606]. Through these mechanisms CD226 is required in vivo for tumor immunosurveillance across transplant, carcinogen-induced, and myeloma models, for NK- and monocyte-dependent control of cytomegalovirus, and for diverse inflammatory functions including monocyte transendothelial migration, macrophage polarization, Treg stability in autoimmunity, and ILC2-dependent airway hyperreactivity [PMID:15136589, PMID:19029379, PMID:25893601, PMID:37625150, PMID:38244725, PMID:27503073]. Receptor abundance is itself regulated: tumor-derived CD155 drives Y319 phosphorylation enabling CBL-B-mediated ubiquitination and proteasomal degradation, and Eomes transcriptionally represses CD226 in CD8+ T cells, with loss of CD226 producing checkpoint-blockade-resistant exhausted T cells [PMID:33053330, PMID:33053331].","teleology":[{"year":1985,"claim":"Before its molecular identity was known, antibody blockade established that this T-lineage activation antigen is functionally required for the differentiation of cytotoxic effectors from precursors.","evidence":"mAb/F(ab')2 blockade in mixed lymphocyte culture with limiting dilution analysis","pmids":["2580933"],"confidence":"Medium","gaps":["Molecular identity of TLiSA1 not yet established","No receptor signaling or ligand defined"]},{"year":1989,"claim":"Linked CD226 surface expression to an upstream regulatory cue by showing TGF-β suppresses it in parallel with cytotoxic effector differentiation.","evidence":"Mixed lymphocyte culture with TGF-β treatment, mAb blockade, and anti-TGF-β rescue","pmids":["2541074"],"confidence":"Medium","gaps":["Mechanism of TGF-β transcriptional control unknown","No molecular cloning yet"]},{"year":1996,"claim":"Defined CD226 as a tyrosine-phosphorylated signal-transducing adhesion molecule required for CTL and NK cytotoxicity, establishing it as more than a passive adhesion antigen.","evidence":"mAb blocking, immunoprecipitation, and cytotoxicity assays","pmids":["8673704"],"confidence":"High","gaps":["Ligand not identified","Specific phosphosites and downstream effectors unknown"]},{"year":1997,"claim":"Molecular cloning unified the T-cell antigen TLiSA1 and platelet antigen PTA1 as a single two-IgV-domain immunoglobulin superfamily protein, resolving its identity and domain architecture.","evidence":"cDNA cloning, peptide sequencing, RT-PCR, flow cytometry, pharmacological perturbation","pmids":["9268302"],"confidence":"High","gaps":["Ligand and signaling partners still unknown","Cytoplasmic motif function uncharacterized"]},{"year":1999,"claim":"Established the functional partnership with LFA-1, showing serine phosphorylation drives the association and Fyn mediates LFA-1-induced CD226 tyrosine phosphorylation, with genetic rescue in LAD NK cells.","evidence":"Reciprocal co-IP, genetic reconstitution of LFA-1 in LAD patient NK cells, phosphorylation assays","pmids":["10591186"],"confidence":"High","gaps":["Precise serine residue not yet mapped","Downstream signaling cascade undefined"]},{"year":2003,"claim":"Mapped the critical Y322 residue for adhesive and costimulatory function across platelet adhesion and naive T cell costimulation, and localized the LFA-1/CD226/Fyn complex to the immunological synapse.","evidence":"Y322F site-directed mutagenesis, lentiviral transduction, adhesion and T cell proliferation/Th1 differentiation assays, synapse imaging","pmids":["12847109","14676297"],"confidence":"High","gaps":["Adaptor recruited by phospho-Y322 not yet identified","Distinction between adhesion and signaling functions incomplete"]},{"year":2004,"claim":"Identified CD155 (PVR) and CD112 (Nectin-2) as the activating ligands and demonstrated cooperation with LFA-1 in triggering cytotoxicity, and showed CD226-PVR governs the diapedesis step of monocyte transendothelial migration.","evidence":"Ectopic ligand expression, cytotoxicity and mAb blocking, soluble DNAM-1-Fc binding, transmigration assays with confocal microscopy","pmids":["15039383","15136589"],"confidence":"High","gaps":["Relative ligand affinities not quantified","Structural basis of binding unknown"]},{"year":2005,"claim":"Consolidated ligand identification by mass spectrometry, defined S329 as the serine site enabling LFA-1-dependent lipid raft recruitment, and demonstrated in vivo tumor rejection requiring CD8+ T, NK cells, and DNAM-1+ dendritic cells.","evidence":"Mass spectrometry, reciprocal chimeric staining, S329 mutagenesis with lipid raft fractionation, in vivo ligand-transduced tumor rejection with depletion/KO","pmids":["15607800","15684041","16249389","15752754"],"confidence":"High","gaps":["Ordering of serine/raft/tyrosine phosphorylation events not fully resolved","Cell-intrinsic vs APC contributions of CD226 not separated"]},{"year":2006,"claim":"Established mechanistic ordering by showing serine phosphorylation induces LFA-1 association which then carries CD226 into rafts, and extended CD226 function to mast cell degranulation synergy with FcεRI.","evidence":"LFA-1-deficient T cells with lipid raft fractionation, mast cell/eosinophil co-culture with pharmacological signaling inhibition","pmids":["16636013","16831868"],"confidence":"High","gaps":["Kinase responsible for serine phosphorylation unidentified","Mast cell pathway relies on inhibitor specificity"]},{"year":2008,"claim":"Germline knockout established CD226 as an in vivo tumor immunosurveillance receptor and defined its context-specific requirement: costimulation for CD8+ T cells with nonprofessional APCs, and elimination of tumors lacking other NK-activating ligands.","evidence":"DNAM-1 KO mice in tumor transplant and carcinogen models, in vitro cytotoxicity and costimulation assays across APC types","pmids":["19029379","19029380"],"confidence":"High","gaps":["Downstream transcriptional effectors not yet defined","Cytoplasmic signaling motif still uncharacterized at the residue level"]},{"year":2012,"claim":"Defined the competitive relationship with the inhibitory receptor TIGIT, showing TIGIT suppresses T cells partly by displacing CD226 from shared CD155.","evidence":"TIGIT shRNA knockdown, agonist/blocking mAb epistasis, cytokine and transcription factor measurement","pmids":["22427644"],"confidence":"High","gaps":["Relative ligand affinities of CD226 vs TIGIT not quantified here","T cell-intrinsic TIGIT mechanism not fully separated"]},{"year":2015,"claim":"Delineated the cytoplasmic ITT-like motif signaling cascade (Grb2/Vav-1/PI3K/PLCγ1/Erk/Akt) driving cytotoxicity, and showed CD226 marks functionally distinct NK subsets and limits multiple myeloma.","evidence":"ITT-motif mutagenesis with Grb2 co-IP and downstream signaling assays, NK transcriptional profiling, Vk*MYC myeloma mouse model with depletion/blockade","pmids":["26552706","25818301","25825444","25893601"],"confidence":"High","gaps":["Transcription factor terminus of the signaling cascade not yet identified","Link between adhesion-only and signaling functions partly unresolved"]},{"year":2018,"claim":"Provided the structural basis for ligand binding via tandem D1–D2 IgV domains, identified FOXO1 inactivation as a terminal signaling output, and established CD226 as an antagonist of TIGIT+ Treg suppressive function.","evidence":"Crystal structure with domain-deletion binding assays, CD226 KO with FOXO1 phosphorylation and pharmacological inhibition, Treg suppression assays in melanoma","pmids":["30591568","30504141","30046006"],"confidence":"High","gaps":["Kinase linking CD226 to FOXO1 phosphorylation not pinpointed","Treg-disrupting mechanism partly correlative"]},{"year":2019,"claim":"Extended CD226 costimulatory function to small peritoneal macrophages as antigen-presenting cells required for CD4+ T cell priming.","evidence":"DNAM-1 KO mice, co-culture antigen presentation, adoptive SPM transfer, in vivo immunization","pmids":["30315271"],"confidence":"High","gaps":["Molecular signaling of CD226 within macrophages not defined here","Ligand source during priming unspecified"]},{"year":2020,"claim":"Revealed multilayered regulation of CD226 abundance and competitive ligand binding: Y319-driven CBL-B ubiquitination/degradation, Eomes-mediated transcriptional repression, preferential competition for soluble CD155 over TIGIT/CD96, and TIGIT-blockade-induced Y322 phosphorylation, all shaping checkpoint blockade outcomes; also defined macrophage polarization roles.","evidence":"Y319 mutagenesis with CBL-B co-IP and proteasomal inhibition, Eomes manipulation with Cd226-/- mice and anti-PD-1, receptor-specific KO mice with sCD155 and affinity measurements, phospho-Y322 analysis, macrophage polarization in MI model","pmids":["33053330","33053331","32040157","32265229","32104514"],"confidence":"High","gaps":["Integration of degradation and transcriptional control not unified","Macrophage CD226 ligand and signaling partly defined"]},{"year":2021,"claim":"Showed CD226 can regulate Treg function purely by sequestering CD155 from TIGIT—without CD226 intracellular signaling—thereby controlling the Akt-mTORC1/Foxp3 axis and Treg stability, and defined PPAR-γ downstream of CD226 in macrophages.","evidence":"DNAM-1-deficient Treg transfer in GvHD, Akt-mTORC1/Foxp3 assays, adipose macrophage KO with VAV1/AKT/FOXO1 phosphorylation and PPAR-γ inhibitor rescue","pmids":["34011606","34823548"],"confidence":"High","gaps":["Generalizability of signaling-independent ligand competition across cell types unclear","PPAR-γ link relies on inhibitor rescue in single lab"]},{"year":2022,"claim":"Defined a stage-dependent requirement for CD226 in human Tfh differentiation, strong early and dispensable/inhibitory at the mature GC-Tfh stage.","evidence":"CD155-expressing artificial APC co-culture with anti-CD226 blockade, proliferation and Tfh marker assays","pmids":["35273617"],"confidence":"Medium","gaps":["Blocking-antibody-based, single lab","Intracellular signaling driving stage-specific effects not mapped"]},{"year":2023,"claim":"Demonstrated via Treg-specific conditional deletion that the CD226/TIGIT balance on Tregs governs Treg stability and autoimmune diabetes incidence.","evidence":"Foxp3-Cre Treg-specific Cd226 KO in NOD mice, diabetes monitoring, TIGIT-Fc functional validation","pmids":["37625150"],"confidence":"High","gaps":["Mechanistic coupling to systemic autoimmunity incomplete","Single disease model"]},{"year":2024,"claim":"Extended CD226 function to innate lymphoid cells, showing inducible CD226 enhances ILC2 effector function via PI3K/AKT and MAPK signaling and drives airway hyperreactivity.","evidence":"Anti-CD226 antibody, RNA-seq of sorted ILC2s, PI3K/AKT and MAPK assays, IL-33 and Alternaria AHR models in WT and Rag2-/- mice","pmids":["38244725"],"confidence":"High","gaps":["ILC2 ligand source in airway not defined","Metabolic reprogramming mechanism incompletely mapped"]},{"year":null,"claim":"How the distinct CD226 phosphosites (Y319 degradation, Y322 signaling, S329 LFA-1/raft recruitment) are coordinately balanced to determine activation versus receptor turnover across cell types, and the kinase directly linking CD226 to FOXO1, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model integrating signaling vs degradation phosphosites","Direct CD226-to-FOXO1 kinase unidentified","Structural model of full signaling complex with LFA-1 lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0,5,7]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[18,23]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[18,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[17,31,28]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[18]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,15,18]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[18,23,35]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[15,21,37]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[26]}],"complexes":[],"partners":["PVR","NECTIN2","ITGAL","FYN","GRB2","CBL","TIGIT"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15762","full_name":"CD226 antigen","aliases":["DNAX accessory molecule 1","DNAM-1"],"length_aa":336,"mass_kda":38.6,"function":"Cell surface receptor that plays an important role in the immune system, particularly in intercellular adhesion, lymphocyte signaling, cytotoxicity and lymphokine secretion mediated by cytotoxic T-cells and NK cells (PubMed:8673704, PubMed:9712030). Functions as a costimulatory receptor upon recognition of target cells, such as virus-infected or tumor cells. Upon binding to its ligands PVR/CD155 or NECTIN2/CD112 on target cells, promotes the cytotoxic activity of NK cells and CTLs, enhancing their ability to kill these cells (PubMed:26755705, PubMed:31253644, PubMed:30591568). Mechanistically, phosphorylation by Src kinases such as LYN of FYN, enables binding to adapter GRB2, leading to activation of VAV1, PI3K and PLCG1. Promotes also activation of kinases ERK and AKT, as well as calcium fluxes (By similarity)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q15762/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CD226","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CD226","total_profiled":1310},"omim":[{"mim_id":"612859","title":"T-CELL IMMUNORECEPTOR WITH IMMUNOGLOBULIN AND ITIM DOMAINS; TIGIT","url":"https://www.omim.org/entry/612859"},{"mim_id":"610436","title":"ROTATIN; RTTN","url":"https://www.omim.org/entry/610436"},{"mim_id":"605397","title":"CD226 ANTIGEN; CD226","url":"https://www.omim.org/entry/605397"},{"mim_id":"603352","title":"BACULOVIRAL IAP REPEAT-CONTAINING PROTEIN 5; BIRC5","url":"https://www.omim.org/entry/603352"},{"mim_id":"600798","title":"NECTIN CELL ADHESION MOLECULE 2; NECTIN2","url":"https://www.omim.org/entry/600798"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Centriolar satellite","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":19.0},{"tissue":"parathyroid gland","ntpm":58.7}],"url":"https://www.proteinatlas.org/search/CD226"},"hgnc":{"alias_symbol":["DNAM-1","DNAM1","PTA1","TLiSA1"],"prev_symbol":[]},"alphafold":{"accession":"Q15762","domains":[{"cath_id":"2.60.40.10","chopping":"4-130","consensus_level":"high","plddt":88.1258,"start":4,"end":130},{"cath_id":"2.60.40.10","chopping":"139-241","consensus_level":"high","plddt":92.1644,"start":139,"end":241}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15762","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15762-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15762-F1-predicted_aligned_error_v6.png","plddt_mean":82.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD226","jax_strain_url":"https://www.jax.org/strain/search?query=CD226"},"sequence":{"accession":"Q15762","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15762.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15762/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15762"}},"corpus_meta":[{"pmid":"8673704","id":"PMC_8673704","title":"DNAM-1, 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domains that is tyrosine-phosphorylated upon activation and functions as a signal-transducing adhesion molecule participating in primary adhesion during CTL-mediated cytotoxicity. Anti-DNAM-1 mAb inhibits T and NK cell-mediated cytotoxicity and blocks cytokine production by alloantigen-specific T cells.\",\n      \"method\": \"Monoclonal antibody blocking, immunoprecipitation, signal transduction analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — original characterization with multiple orthogonal methods (mAb inhibition, phosphorylation assay, cytotoxicity assay), replicated across subsequent studies\",\n      \"pmids\": [\"8673704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"TLiSA1 (CD226) is a T lineage-specific activation antigen whose antibody-mediated blockade from the beginning of mixed lymphocyte culture inhibits the induction of both CTL and anomalous killer (AK) cells from precursors, reducing CTL frequency and greatly reducing AK cell frequency under limiting dilution conditions, indicating a role in differentiation of cytotoxic effectors.\",\n      \"method\": \"Mixed lymphocyte culture with mAb blockade, limiting dilution analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional blockade with mAb and F(ab')2 fragments, limiting dilution confirmation, single lab\",\n      \"pmids\": [\"2580933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"TGF-β suppresses TLiSA1 (CD226) surface expression and inhibits CTL and LAK cell differentiation in a dose-dependent manner; antibody to TGF-β reverses both effects, and anti-TLiSA1 antibody mimics TGF-β in inhibiting cytotoxic effector differentiation, linking TLiSA1 expression to the differentiation process.\",\n      \"method\": \"Mixed lymphocyte culture with TGF-β treatment, mAb blocking, anti-TGF-β rescue\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cytokine and antibody perturbation with rescue experiment, single lab\",\n      \"pmids\": [\"2541074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"TLiSA1/PTA1 (CD226) was cloned and identified as a novel immunoglobulin superfamily member with two V-set domains only. Identity between the T cell activation antigen TLiSA1 and platelet antigen PTA1 was established by immunological criteria, internal peptide sequencing, and RT-PCR of platelet transcript. Phorbol ester stimulates mRNA and surface expression, while combined phorbol ester and ionophore suppresses expression via a calcineurin-independent pathway.\",\n      \"method\": \"cDNA cloning, peptide sequencing, RT-PCR, flow cytometry, pharmacological perturbation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cDNA cloning with structural determination, peptide sequence confirmation, multiple orthogonal methods\",\n      \"pmids\": [\"9268302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"LFA-1 physically associates with DNAM-1 in NK cells and anti-CD3-stimulated T cells, and serine phosphorylation of DNAM-1 plays a critical role in this association. Cross-linking of LFA-1 induces tyrosine phosphorylation of DNAM-1, for which the Fyn protein tyrosine kinase is responsible. DNAM-1-mediated cytotoxicity is defective in LFA-1-deficient (LAD syndrome) NK cells and is restored by genetic reconstitution of LFA-1 expression.\",\n      \"method\": \"Co-immunoprecipitation, genetic reconstitution, phosphorylation assays, cytotoxicity assays with LAD patient NK cells\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reciprocal co-IP, phosphorylation mapping, genetic rescue experiment, multiple orthogonal approaches\",\n      \"pmids\": [\"10591186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD226 mediates platelet adhesion to vascular endothelial cells; thrombin-activated (but not resting) platelets bind to intact HUVEC via CD226, and thrombin-induced platelet activation induces tyrosine phosphorylation of CD226. Mutant transfectant experiments demonstrated that tyrosine at residue 322 is critical for CD226 adhesive function. CD226 is also expressed on megakaryocytic cells and mediates their adhesion to HUVEC.\",\n      \"method\": \"mAb blocking adhesion assay, site-directed mutagenesis (Y322 mutant transfectants), phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis of specific residue combined with functional adhesion assay, phosphorylation measurement, multiple cell types\",\n      \"pmids\": [\"12847109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Upon TCR antigen recognition, LFA-1 physically associates with CD226 (DNAM-1) and the protein tyrosine kinase Fyn. Lentiviral expression of mutant Y322F CD226 in naive CD4+ and CD8+ T cells inhibited LFA-1-mediated costimulatory signals for naive T cell proliferation and IL-12-independent Th1 differentiation. LFA-1, CD226, and Fyn are polarized at the immunological synapse upon CD3 stimulation. Lipid raft disruption inhibits LFA-1 costimulatory signaling in CD4+ but not CD8+ T cells.\",\n      \"method\": \"Lentiviral mutant transduction (Y322F), T cell proliferation assay, Th1 differentiation assay, immunological synapse imaging, lipid raft disruption\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — site-directed mutagenesis with lentiviral rescue, multiple functional readouts, co-IP of complex, imaging\",\n      \"pmids\": [\"14676297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human CD155 (PVR) and CD112 (Nectin-2/PRR-2) were identified as ligands for human CD226. Ectopic expression of CD155 and/or CD112 on mouse BW5147 T cells increased their susceptibility to IL-2-activated T and NK cell-mediated cytotoxicity, which was specifically inhibited by anti-CD226 mAb. Ligation of CD226 and LFA-1 cooperates in triggering cytotoxicity and cytokine secretion.\",\n      \"method\": \"Ectopic expression/transfection, cytotoxicity assay, mAb blocking, binding affinity measurement\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reconstitution via transfection, specific inhibition by anti-CD226 mAb, replicated across multiple labs\",\n      \"pmids\": [\"15039383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DNAM-1 (CD226) regulates monocyte transendothelial migration; PVR (CD155) is expressed at endothelial cell junctions and is the major DNAM-1 ligand on endothelial cells (Nectin-2 contributes less). Anti-DNAM-1 and anti-PVR mAbs blocked monocyte transmigration in vitro, arresting monocytes at the apical surface over intercellular junctions, indicating DNAM-1-PVR interaction occurs during the diapedesis step.\",\n      \"method\": \"Soluble DNAM-1-Fc binding assay, mAb blocking, transendothelial migration assay, confocal microscopy\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — soluble receptor binding with specific antibody competition, functional transmigration blockade with stage-specific phenotype, multiple orthogonal approaches\",\n      \"pmids\": [\"15136589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PVR and Nectin-2 were confirmed as DNAM-1 ligands by mass spectrometry identification of purified proteins and by demonstrating mutual staining between PVR-Fc/Nectin-2-Fc chimeric molecules and DNAM-1-expressing cells and vice versa. DNAM-1/PVR or Nectin-2 interaction enhanced NK-mediated lysis of tumor cells.\",\n      \"method\": \"Tryptic digestion and mass spectrometry, chimeric protein staining, cytotoxicity assay with mAb blocking\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mass spectrometry identification, reciprocal binding validation, functional cytotoxicity assay, replicated\",\n      \"pmids\": [\"15607800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"DNAM-1 ligand-transduced RMA tumor cells were rejected in syngeneic mice in a CD8+ T cell- and NK cell-dependent manner generating CD8+ memory CTL. DNAM-1 is expressed on CD8α+ dendritic cells, and cross-linking DNAM-1 on these DCs induced their maturation and Th1 cell priming. Tumor rejection was abrogated in CD4+ T cell-depleted and MHC class II-deficient mice.\",\n      \"method\": \"Ligand transduction, in vivo tumor rejection assay, DC maturation assay, T cell depletion, KO mouse model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple in vivo genetic and depletion approaches, DC functional assay, multiple cell type contributions defined\",\n      \"pmids\": [\"16249389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Serine phosphorylation at residue 329 (S329) of DNAM-1 is required for LFA-1-dependent lipid raft recruitment of DNAM-1. Wild-type but not S329 mutant DNAM-1 associates with lipid rafts at peripheral supra-molecular activation clusters, and lipid raft association is necessary for tyrosine phosphorylation of DNAM-1, which is essential for LFA-1-mediated costimulatory signaling for naive T cell proliferation and differentiation.\",\n      \"method\": \"Site-directed mutagenesis (S329), lipid raft fractionation, immunological synapse imaging, T cell functional assays\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — specific residue mutagenesis, lipid raft biochemical fractionation, functional consequence demonstrated\",\n      \"pmids\": [\"15684041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Murine DNAM-1 and its PVR homologue were identified; they physically bind each other and ligand binding of murine DNAM-1 induces a costimulatory signal in antigen-specific CD8+ T cells.\",\n      \"method\": \"Binding assays, T cell costimulation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed, functional costimulation assay, single lab\",\n      \"pmids\": [\"15752754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Human mast cells and eosinophils express DNAM-1 (CD226) and its ligand Nectin-2 (CD112), respectively. CD226 engagement on mast cells synergizes with FcεRI and augments degranulation through a pathway involving Fyn, LAT, PLCγ2, and CD18. Blocking CD112 on eosinophils normalized the hyperactivity of IgE-activated mast cells co-cultured with eosinophils. This pathway is completely inhibited by linking IgE with IRp60 using a bispecific antibody.\",\n      \"method\": \"mAb blocking, co-culture assay, pharmacological inhibition of signaling intermediates, bispecific antibody\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined signaling pathway with pharmacological inhibitors, co-culture system, single lab\",\n      \"pmids\": [\"16831868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Lipid raft recruitment of DNAM-1 in CD4+ T cells depends on LFA-1 expression; in LFA-1-deficient CD4+ T cells, TCR-mediated serine phosphorylation of DNAM-1 occurs but DNAM-1 does not associate with lipid rafts, indicating serine phosphorylation of DNAM-1 primarily induces physical association with LFA-1, which then carries DNAM-1 into lipid rafts.\",\n      \"method\": \"LFA-1-deficient mouse T cells, lipid raft fractionation, phosphorylation assay\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genetic knockout of LFA-1 combined with biochemical fractionation, mechanistic ordering of events established\",\n      \"pmids\": [\"16636013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNAM-1-deficient mice show accelerated tumor development and mortality after transplantation of DNAM-1 ligand (CD155)-expressing Meth A fibrosarcoma cells. DNAM-1-deficient CTL and NK cells show significantly reduced cytotoxic activity against DNAM-1 ligand-expressing tumor cells in vitro. DNAM-1-deficient mice also develop significantly more fibrosarcoma and papilloma in response to chemical carcinogens MCA and DMBA, demonstrating an in vivo tumor immunosurveillance role.\",\n      \"method\": \"DNAM-1 knockout mice, tumor transplantation model, chemical carcinogen model, in vitro cytotoxicity assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — germline KO with multiple tumor models (transplant and carcinogen-induced), replicated across labs\",\n      \"pmids\": [\"19029379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CD8+ T cells require DNAM-1 for co-stimulation when antigen is presented by nonprofessional APCs but not dendritic cells. NK cells require DNAM-1 for elimination of tumor cells with paucity of other NK-activating ligands. DNAM-1-deficient mice generated from knockout model demonstrated these cell-type and context-specific requirements.\",\n      \"method\": \"DNAM-1 knockout mice, in vitro T cell co-stimulation assay, in vitro NK cytotoxicity assay, DC vs. non-professional APC comparison\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined cellular phenotype, independent lab from PMID 19029379, multiple functional readouts\",\n      \"pmids\": [\"19029380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TIGIT exerts immunosuppressive effects on CD4+ T cells by competing with CD226 for binding to CD155. Knockdown of TIGIT by shRNA increased T-bet and IFN-γ expression; this increase was overcome by blocking CD226 signaling, demonstrating that TIGIT inhibits T cell function partly by displacing CD226 from its CD155 ligand. TIGIT also directly inhibits T cells in a T cell-intrinsic manner.\",\n      \"method\": \"shRNA knockdown of TIGIT, anti-TIGIT agonist mAb, anti-CD226 blocking mAb, cytokine/transcription factor measurement\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockdown combined with receptor blockade epistasis, multiple orthogonal methods, functional rescue\",\n      \"pmids\": [\"22427644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DNAM-1 controls NK cell-mediated cytotoxicity and cytokine production via a conserved tyrosine- and asparagine-based ITT-like motif in its cytoplasmic domain. Upon Src kinase-mediated phosphorylation of this motif, DNAM-1 binds adaptor Grb2, leading to activation of Vav-1, PI3K, and PLCγ1, as well as Erk and Akt kinases and calcium flux. DNAM-1 signaling also enhances cytotoxicity by increasing actin polymerization and granule polarization. DNAM-1-mediated adhesion is signal-independent and insufficient alone for cytotoxicity.\",\n      \"method\": \"Site-directed mutagenesis of ITT-like motif, co-immunoprecipitation of Grb2, downstream signaling assays (Erk, Akt, calcium flux), actin polymerization assay, granule polarization assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis of specific signaling motif, co-IP of downstream adaptor Grb2, multiple downstream effectors characterized with orthogonal methods\",\n      \"pmids\": [\"26552706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DNAM-1 expression identifies two functionally distinct NK cell subsets: DNAM-1+ NK cells produce high levels of inflammatory cytokines, have enhanced IL-15 signaling, and proliferate vigorously; DNAM-1- NK cells differentiate from DNAM-1+ cells, express more NK receptor-related genes, and produce higher levels of MIP1 chemokines.\",\n      \"method\": \"Transcriptional profiling, functional cytokine production assay, proliferation assay, IL-15 signaling assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptional profiling combined with functional studies, single lab\",\n      \"pmids\": [\"25818301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In educated NK cells, upon target cell recognition, the conformational state of LFA-1 changes and both active LFA-1 and DNAM-1 rapidly colocalize at the immune synapse. DNAM-1 expression correlates with the quantity and quality of inhibitory input by HLA class I-specific KIRs and CD94/NKG2A, and with the magnitude of functional responses.\",\n      \"method\": \"Flow cytometry, live imaging, functional cytotoxicity correlation analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct imaging of immune synapse localization with functional correlation, single lab\",\n      \"pmids\": [\"25825444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD226 is required for immunosurveillance and therapy of multiple myeloma; CD226 limits spontaneous MM development in Vk*MYC transgenic mice. The anti-myeloma immune response is mediated by both NK and CD8+ T cells through perforin and IFN-γ pathways. CD226 expression is also required for optimal antimyeloma efficacy of cyclophosphamide and bortezomib.\",\n      \"method\": \"Cd226 mutant mice crossed with Vk*MYC MM model, NK/T cell depletion, perforin/IFN-γ blockade, drug treatment\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model with long-term surveillance study, mechanistic pathway defined by depletion/blockade, drug efficacy dependence\",\n      \"pmids\": [\"25893601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD226 binding to CD155 is mediated by a unique side-by-side arrangement of two tandem IgV domains (D1 and D2). Crystal structure of mCD226-ecto revealed that D1 forms the conserved binding interface with CD155-D1, while D2 provides structural support and forms direct interactions with CD155. Deletion of D2 substantially reduced CD226 binding efficacy to CD155.\",\n      \"method\": \"Crystal structure determination (X-ray crystallography), deletion mutant binding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by domain deletion, structural basis for binding established\",\n      \"pmids\": [\"30591568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD226 regulates NK cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1. Engagement of CD155 ligand results in phosphorylation of FOXO1 downstream of CD226. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses.\",\n      \"method\": \"CD226-deficient mice, gene expression analysis, in vitro cytotoxicity/stimulation assay, FOXO1 phosphorylation assay, pharmacological inhibitors\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genetic KO combined with pharmacological inhibition and phosphorylation assays, multiple orthogonal approaches defining a specific transcription factor as downstream effector\",\n      \"pmids\": [\"30504141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD226 opposes TIGIT to disrupt Treg suppressive function in melanoma; TIGIT+ Tregs are highly suppressive and stable while CD226 disrupts Treg suppression and stability. A high TIGIT/CD226 ratio in Tregs correlates with increased Treg frequencies in tumors.\",\n      \"method\": \"Flow cytometry of Tregs from melanoma patients and tumors, functional suppression assay, mAb blocking of TIGIT and CD226\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor blocking functional assay in patient samples, single lab, correlation plus functional intervention\",\n      \"pmids\": [\"30046006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DNAM-1 on small peritoneal macrophages (SPMs) is required for CD4+ T cell co-stimulation; SPMs but not large peritoneal macrophages (LPMs) express DNAM-1 and have antigen-presenting capacity. DNAM-1 deficiency on SPMs or blockade of DNAM-1/ligand interaction impaired CD4+ T cell priming. Wild-type but not DNAM-1-deficient SPMs enhanced T and B cell responses in vivo upon adoptive transfer.\",\n      \"method\": \"DNAM-1 KO mice, co-culture antigen presentation assay, adoptive SPM transfer, in vivo immunization\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO combined with adoptive transfer, multiple in vitro and in vivo functional readouts\",\n      \"pmids\": [\"30315271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tumor-derived CD155 initiates phosphorylation of CD226 at tyrosine 319 (Y319) by Src kinases, which enables ubiquitination of CD226 by the E3 ligase CBL-B, followed by internalization and proteasomal degradation of CD226. Mutation of Y319 led to increased CD226 surface expression, enhanced anti-tumor immunity, and improved immune checkpoint blockade efficacy.\",\n      \"method\": \"Y319 mutagenesis, phosphorylation assay, CBL-B co-immunoprecipitation, proteasomal inhibition, in vivo tumor models\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — specific residue mutagenesis, identification of E3 ligase CBL-B, proteasomal degradation pathway validated, in vivo functional consequence\",\n      \"pmids\": [\"33053330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of CD226 expression on CD8+ T cells is driven by the transcriptional regulator Eomesodermin (Eomes) through an antigen-specific mechanism. CD226-negative CD8+ T cells exhibit reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program. CD226-deficient CD8+ TILs fail to respond to anti-PD-1, and immune checkpoint blockade efficacy was hampered in Cd226-/- mice.\",\n      \"method\": \"Transcriptome analysis, Eomes overexpression/knockdown, Cd226-/- mice, anti-PD-1 treatment, LFA-1 activation assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined transcriptional mechanism, multiple functional readouts, published alongside companion paper\",\n      \"pmids\": [\"33053331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Soluble CD155 (sCD155) secreted by tumor cells inhibits DNAM-1-mediated NK cell degranulation and promotes lung colonization of B16/BL6 melanoma; in vivo the effect required DNAM-1 (not TIGIT or CD96). DNAM-1 has greater affinity than TIGIT and CD96 for sCD155, indicating preferential competitive binding.\",\n      \"method\": \"sCD155-expressing tumor injection in KO mice (DNAM-1, TIGIT, CD96), in vitro degranulation assay, binding affinity measurement\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple receptor-specific KO mice, in vitro and in vivo complementary approaches, binding affinity data\",\n      \"pmids\": [\"32040157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Anti-TIGIT treatment selectively promotes CD226 phosphorylation at tyrosine 322 in CD226hiCD8+ T cells, and CD226 agonist antibody augments the effect of TIGIT blockade on CD8+ T cell responses. CD226loCD8+ T cells accumulate at the tumor site with exhausted phenotype.\",\n      \"method\": \"Flow cytometry, phospho-Y322 analysis after anti-TIGIT treatment, CD226 agonist antibody functional assay\",\n      \"journal\": \"Cancer immunology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific phosphorylation site measured in response to TIGIT blockade, functional agonist assay, single lab\",\n      \"pmids\": [\"32265229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD226 deletion in mice improves post-myocardial infarction healing by favoring macrophage M2 polarization and suppressing M1 polarization. In vitro, CD226 deletion in bone marrow-derived macrophages potentiated M2 and suppressed M1 polarization. CD226 deletion also restrained inflammatory monocyte mobilization from spleen to blood.\",\n      \"method\": \"CD226 KO mice with coronary ligation model, bone marrow-derived macrophage polarization assay, flow cytometry of monocyte subsets\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with in vivo and in vitro macrophage polarization assays, single lab\",\n      \"pmids\": [\"32104514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DNAM-1 competes with TIGIT in binding to their common ligand CD155, thereby regulating TIGIT signaling to down-regulate Treg cell function without requiring DNAM-1-mediated intracellular signaling. DNAM-1 deficiency augments TIGIT signaling, which inhibits Akt-mTORC1 pathway activation, maintaining Foxp3 expression and Treg function under inflammatory conditions. Transfer of DNAM-1-deficient Treg cells ameliorated graft-versus-host disease better than wild-type Tregs.\",\n      \"method\": \"DNAM-1-deficient Treg transfer, GvHD model, Akt-mTORC1 signaling assay, Foxp3 stability assay, TIGIT signaling measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO Treg transfer with GvHD functional readout, defined signaling pathway (Akt-mTORC1/Foxp3), mechanistic ordering established\",\n      \"pmids\": [\"34011606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CD226 deficiency on adipose tissue macrophages (ATMs) decreases phosphorylation of VAV1, AKT, and FOXO1 and upregulates PPAR-γ, thereby inhibiting macrophage M1 polarization. Administration of a PPAR-γ inhibitor restored M1 phenotype in CD226KO ATMs, placing PPAR-γ downstream of CD226 signaling in macrophage polarization.\",\n      \"method\": \"CD226 KO mice, HFD obesity model, phosphorylation assays (VAV1, AKT, FOXO1), PPAR-γ inhibitor rescue experiment\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with pharmacological rescue defining PPAR-γ as downstream effector, single lab\",\n      \"pmids\": [\"34823548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CD226 signals promote the early phase of human Tfh cell differentiation; blocking CD226 potently inhibited proliferation and Tfh marker expression of naive CD4+ T cells and Tfh precursors stimulated with CD155-expressing APCs. In contrast, mature GC-Tfh cells require only weak CD226 signals, and attenuating CD226 rather increased CXCR5, ICOS, and IL-21 expression, indicating the requirement for CD226 wanes as Tfh cells mature.\",\n      \"method\": \"CD155-expressing artificial APC co-culture, anti-CD226 blocking antibody, proliferation assay, Tfh marker expression assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — blocking antibody functional assay with defined differentiation stages, single lab\",\n      \"pmids\": [\"35273617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Treg-specific Cd226 deletion (TregΔCd226 mice) decreased insulitis and diabetes incidence in NOD mice. CD226-deficient pancreatic Tregs had increased TIGIT expression. NOD splenocytes treated with TIGIT-Fc fusion protein showed reduced T cell proliferation and IFN-γ production, indicating CD226/TIGIT imbalance on Tregs contributes to Treg instability.\",\n      \"method\": \"Conditional Treg-specific KO (Foxp3-Cre), diabetes incidence monitoring, TIGIT-Fc fusion protein treatment, proliferation and cytokine assays\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional KO with disease endpoint, TIGIT-Fc functional validation, multiple methods\",\n      \"pmids\": [\"37625150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CD226 is inducibly expressed in activated ILC2s and enhances their cytokine secretion and effector functions. Mechanistically, CD226 alters intracellular metabolism and enhances PI3K/AKT and MAPK signaling pathways in ILC2s. Blocking CD226 ameliorates ILC2-dependent airway hyperreactivity in IL-33 and Alternaria alternata-induced models.\",\n      \"method\": \"Anti-CD226 antibody treatment, RNA sequencing of sorted ILC2s, PI3K/AKT and MAPK signaling assay, in vivo AHR mouse models (WT and Rag2-/- mice)\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNA sequencing, defined signaling pathway, in vivo functional assay in multiple models, multiple orthogonal approaches\",\n      \"pmids\": [\"38244725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The alphaherpesvirus gD glycoprotein causes degradation and down-regulation of CD112 (a DNAM-1 ligand), reducing DNAM-1 binding to infected cells and suppressing DNAM-1-mediated NK cell degranulation and lysis of PRV- or HSV-2-infected cells.\",\n      \"method\": \"Viral infection/transfection with gD expression, flow cytometry of CD112, NK degranulation assay, cytotoxicity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined viral protein responsible, CD112 degradation linked to impaired DNAM-1 binding and NK function, single lab\",\n      \"pmids\": [\"25352670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mouse CMV m20.1 protein retains PVR (DNAM-1 ligand) in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking m20.1 was attenuated in normal but not DNAM-1 KO mice. This attenuation was partially reversed by NK cell depletion and abolished by simultaneous depletion of mononuclear phagocytes, indicating that inflammatory monocytes together with NK cells control CMV through the DNAM-1-PVR pathway.\",\n      \"method\": \"MCMV m20.1 deletion mutant, DNAM-1 KO mice, cell depletion, iNOS inhibition, CCL2 blocking\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — viral KO mutant combined with host KO mice and cell depletion, specific viral immune evasion mechanism identified\",\n      \"pmids\": [\"27503073\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD226 (DNAM-1) is a two-IgV-domain immunoglobulin superfamily transmembrane receptor that binds CD155 (PVR) and CD112 (Nectin-2) via its D1–D2 ectodomain in a side-by-side arrangement; upon ligand engagement it undergoes Src kinase-mediated phosphorylation at Y319 (enabling CBL-B-mediated ubiquitination and proteasomal degradation) and serine phosphorylation at S329 (enabling LFA-1 association and lipid raft recruitment), while signaling through a cytoplasmic ITT-like motif that recruits Grb2 and activates Vav-1, PI3K, PLCγ1, Erk, and Akt to drive actin polymerization, granule polarization, cytokine production, and FOXO1 inactivation; it competes with the inhibitory receptor TIGIT for shared ligands to promote effector lymphocyte (NK, CD8+ T, and Treg) function, and is required in vivo for tumor immunosurveillance, control of viral infections, and regulation of inflammatory responses including monocyte transendothelial migration and ILC2-dependent airway hyperreactivity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CD226 (DNAM-1) is an immunoglobulin-superfamily transmembrane glycoprotein with two V-set domains that functions as an activating adhesion and signaling receptor central to cytotoxic lymphocyte function and immunosurveillance [#0, #3]. It engages the nectin-family ligands CD155 (PVR) and CD112 (Nectin-2) through a side-by-side tandem D1\\u2013D2 IgV arrangement, in which D1 forms the conserved interface with CD155 while D2 provides structural support and direct contacts required for high-affinity binding [#7, #22]. Ligand engagement triggers Src-family kinase-mediated tyrosine phosphorylation of a cytoplasmic ITT-like motif (residue Y322/Y319 critical for adhesive and signaling function) that recruits Grb2 and activates Vav-1, PI3K, PLC\\u03b31, Erk, and Akt, driving actin polymerization, granule polarization, calcium flux, cytokine production, and phosphorylation-mediated inactivation of FOXO1 [#5, #18, #23]. Serine phosphorylation at S329 instead promotes physical association with LFA-1, which carries CD226 into lipid rafts at the immunological synapse; this LFA-1 partnership is required for CD226 tyrosine phosphorylation and costimulatory signaling, and Fyn mediates the bidirectional phosphorylation between the two receptors [#4, #6, #11, #14]. CD226 competes with the inhibitory receptor TIGIT (and outcompetes CD96) for the shared CD155 ligand, and the CD226/TIGIT balance dictates effector versus regulatory outcomes in CD8+ T cells, NK cells, and Tregs [#17, #28, #31]. Through these mechanisms CD226 is required in vivo for tumor immunosurveillance across transplant, carcinogen-induced, and myeloma models, for NK- and monocyte-dependent control of cytomegalovirus, and for diverse inflammatory functions including monocyte transendothelial migration, macrophage polarization, Treg stability in autoimmunity, and ILC2-dependent airway hyperreactivity [#8, #15, #21, #34, #35, #37]. Receptor abundance is itself regulated: tumor-derived CD155 drives Y319 phosphorylation enabling CBL-B-mediated ubiquitination and proteasomal degradation, and Eomes transcriptionally represses CD226 in CD8+ T cells, with loss of CD226 producing checkpoint-blockade-resistant exhausted T cells [#26, #27].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Before its molecular identity was known, antibody blockade established that this T-lineage activation antigen is functionally required for the differentiation of cytotoxic effectors from precursors.\",\n      \"evidence\": \"mAb/F(ab')2 blockade in mixed lymphocyte culture with limiting dilution analysis\",\n      \"pmids\": [\"2580933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity of TLiSA1 not yet established\", \"No receptor signaling or ligand defined\"]\n    },\n    {\n      \"year\": 1989,\n      \"claim\": \"Linked CD226 surface expression to an upstream regulatory cue by showing TGF-\\u03b2 suppresses it in parallel with cytotoxic effector differentiation.\",\n      \"evidence\": \"Mixed lymphocyte culture with TGF-\\u03b2 treatment, mAb blockade, and anti-TGF-\\u03b2 rescue\",\n      \"pmids\": [\"2541074\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of TGF-\\u03b2 transcriptional control unknown\", \"No molecular cloning yet\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Defined CD226 as a tyrosine-phosphorylated signal-transducing adhesion molecule required for CTL and NK cytotoxicity, establishing it as more than a passive adhesion antigen.\",\n      \"evidence\": \"mAb blocking, immunoprecipitation, and cytotoxicity assays\",\n      \"pmids\": [\"8673704\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand not identified\", \"Specific phosphosites and downstream effectors unknown\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Molecular cloning unified the T-cell antigen TLiSA1 and platelet antigen PTA1 as a single two-IgV-domain immunoglobulin superfamily protein, resolving its identity and domain architecture.\",\n      \"evidence\": \"cDNA cloning, peptide sequencing, RT-PCR, flow cytometry, pharmacological perturbation\",\n      \"pmids\": [\"9268302\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand and signaling partners still unknown\", \"Cytoplasmic motif function uncharacterized\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the functional partnership with LFA-1, showing serine phosphorylation drives the association and Fyn mediates LFA-1-induced CD226 tyrosine phosphorylation, with genetic rescue in LAD NK cells.\",\n      \"evidence\": \"Reciprocal co-IP, genetic reconstitution of LFA-1 in LAD patient NK cells, phosphorylation assays\",\n      \"pmids\": [\"10591186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise serine residue not yet mapped\", \"Downstream signaling cascade undefined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped the critical Y322 residue for adhesive and costimulatory function across platelet adhesion and naive T cell costimulation, and localized the LFA-1/CD226/Fyn complex to the immunological synapse.\",\n      \"evidence\": \"Y322F site-directed mutagenesis, lentiviral transduction, adhesion and T cell proliferation/Th1 differentiation assays, synapse imaging\",\n      \"pmids\": [\"12847109\", \"14676297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor recruited by phospho-Y322 not yet identified\", \"Distinction between adhesion and signaling functions incomplete\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified CD155 (PVR) and CD112 (Nectin-2) as the activating ligands and demonstrated cooperation with LFA-1 in triggering cytotoxicity, and showed CD226-PVR governs the diapedesis step of monocyte transendothelial migration.\",\n      \"evidence\": \"Ectopic ligand expression, cytotoxicity and mAb blocking, soluble DNAM-1-Fc binding, transmigration assays with confocal microscopy\",\n      \"pmids\": [\"15039383\", \"15136589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative ligand affinities not quantified\", \"Structural basis of binding unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Consolidated ligand identification by mass spectrometry, defined S329 as the serine site enabling LFA-1-dependent lipid raft recruitment, and demonstrated in vivo tumor rejection requiring CD8+ T, NK cells, and DNAM-1+ dendritic cells.\",\n      \"evidence\": \"Mass spectrometry, reciprocal chimeric staining, S329 mutagenesis with lipid raft fractionation, in vivo ligand-transduced tumor rejection with depletion/KO\",\n      \"pmids\": [\"15607800\", \"15684041\", \"16249389\", \"15752754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ordering of serine/raft/tyrosine phosphorylation events not fully resolved\", \"Cell-intrinsic vs APC contributions of CD226 not separated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established mechanistic ordering by showing serine phosphorylation induces LFA-1 association which then carries CD226 into rafts, and extended CD226 function to mast cell degranulation synergy with Fc\\u03b5RI.\",\n      \"evidence\": \"LFA-1-deficient T cells with lipid raft fractionation, mast cell/eosinophil co-culture with pharmacological signaling inhibition\",\n      \"pmids\": [\"16636013\", \"16831868\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for serine phosphorylation unidentified\", \"Mast cell pathway relies on inhibitor specificity\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Germline knockout established CD226 as an in vivo tumor immunosurveillance receptor and defined its context-specific requirement: costimulation for CD8+ T cells with nonprofessional APCs, and elimination of tumors lacking other NK-activating ligands.\",\n      \"evidence\": \"DNAM-1 KO mice in tumor transplant and carcinogen models, in vitro cytotoxicity and costimulation assays across APC types\",\n      \"pmids\": [\"19029379\", \"19029380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream transcriptional effectors not yet defined\", \"Cytoplasmic signaling motif still uncharacterized at the residue level\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the competitive relationship with the inhibitory receptor TIGIT, showing TIGIT suppresses T cells partly by displacing CD226 from shared CD155.\",\n      \"evidence\": \"TIGIT shRNA knockdown, agonist/blocking mAb epistasis, cytokine and transcription factor measurement\",\n      \"pmids\": [\"22427644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative ligand affinities of CD226 vs TIGIT not quantified here\", \"T cell-intrinsic TIGIT mechanism not fully separated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Delineated the cytoplasmic ITT-like motif signaling cascade (Grb2/Vav-1/PI3K/PLC\\u03b31/Erk/Akt) driving cytotoxicity, and showed CD226 marks functionally distinct NK subsets and limits multiple myeloma.\",\n      \"evidence\": \"ITT-motif mutagenesis with Grb2 co-IP and downstream signaling assays, NK transcriptional profiling, Vk*MYC myeloma mouse model with depletion/blockade\",\n      \"pmids\": [\"26552706\", \"25818301\", \"25825444\", \"25893601\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcription factor terminus of the signaling cascade not yet identified\", \"Link between adhesion-only and signaling functions partly unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided the structural basis for ligand binding via tandem D1\\u2013D2 IgV domains, identified FOXO1 inactivation as a terminal signaling output, and established CD226 as an antagonist of TIGIT+ Treg suppressive function.\",\n      \"evidence\": \"Crystal structure with domain-deletion binding assays, CD226 KO with FOXO1 phosphorylation and pharmacological inhibition, Treg suppression assays in melanoma\",\n      \"pmids\": [\"30591568\", \"30504141\", \"30046006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase linking CD226 to FOXO1 phosphorylation not pinpointed\", \"Treg-disrupting mechanism partly correlative\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended CD226 costimulatory function to small peritoneal macrophages as antigen-presenting cells required for CD4+ T cell priming.\",\n      \"evidence\": \"DNAM-1 KO mice, co-culture antigen presentation, adoptive SPM transfer, in vivo immunization\",\n      \"pmids\": [\"30315271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular signaling of CD226 within macrophages not defined here\", \"Ligand source during priming unspecified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed multilayered regulation of CD226 abundance and competitive ligand binding: Y319-driven CBL-B ubiquitination/degradation, Eomes-mediated transcriptional repression, preferential competition for soluble CD155 over TIGIT/CD96, and TIGIT-blockade-induced Y322 phosphorylation, all shaping checkpoint blockade outcomes; also defined macrophage polarization roles.\",\n      \"evidence\": \"Y319 mutagenesis with CBL-B co-IP and proteasomal inhibition, Eomes manipulation with Cd226-/- mice and anti-PD-1, receptor-specific KO mice with sCD155 and affinity measurements, phospho-Y322 analysis, macrophage polarization in MI model\",\n      \"pmids\": [\"33053330\", \"33053331\", \"32040157\", \"32265229\", \"32104514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of degradation and transcriptional control not unified\", \"Macrophage CD226 ligand and signaling partly defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed CD226 can regulate Treg function purely by sequestering CD155 from TIGIT\\u2014without CD226 intracellular signaling\\u2014thereby controlling the Akt-mTORC1/Foxp3 axis and Treg stability, and defined PPAR-\\u03b3 downstream of CD226 in macrophages.\",\n      \"evidence\": \"DNAM-1-deficient Treg transfer in GvHD, Akt-mTORC1/Foxp3 assays, adipose macrophage KO with VAV1/AKT/FOXO1 phosphorylation and PPAR-\\u03b3 inhibitor rescue\",\n      \"pmids\": [\"34011606\", \"34823548\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generalizability of signaling-independent ligand competition across cell types unclear\", \"PPAR-\\u03b3 link relies on inhibitor rescue in single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a stage-dependent requirement for CD226 in human Tfh differentiation, strong early and dispensable/inhibitory at the mature GC-Tfh stage.\",\n      \"evidence\": \"CD155-expressing artificial APC co-culture with anti-CD226 blockade, proliferation and Tfh marker assays\",\n      \"pmids\": [\"35273617\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Blocking-antibody-based, single lab\", \"Intracellular signaling driving stage-specific effects not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated via Treg-specific conditional deletion that the CD226/TIGIT balance on Tregs governs Treg stability and autoimmune diabetes incidence.\",\n      \"evidence\": \"Foxp3-Cre Treg-specific Cd226 KO in NOD mice, diabetes monitoring, TIGIT-Fc functional validation\",\n      \"pmids\": [\"37625150\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic coupling to systemic autoimmunity incomplete\", \"Single disease model\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended CD226 function to innate lymphoid cells, showing inducible CD226 enhances ILC2 effector function via PI3K/AKT and MAPK signaling and drives airway hyperreactivity.\",\n      \"evidence\": \"Anti-CD226 antibody, RNA-seq of sorted ILC2s, PI3K/AKT and MAPK assays, IL-33 and Alternaria AHR models in WT and Rag2-/- mice\",\n      \"pmids\": [\"38244725\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ILC2 ligand source in airway not defined\", \"Metabolic reprogramming mechanism incompletely mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the distinct CD226 phosphosites (Y319 degradation, Y322 signaling, S329 LFA-1/raft recruitment) are coordinately balanced to determine activation versus receptor turnover across cell types, and the kinase directly linking CD226 to FOXO1, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model integrating signaling vs degradation phosphosites\", \"Direct CD226-to-FOXO1 kinase unidentified\", \"Structural model of full signaling complex with LFA-1 lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 5, 7]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [18, 23]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [18, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [17, 31, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 15, 18]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [18, 23, 35]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15, 21, 37]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [26]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PVR\", \"NECTIN2\", \"ITGAL\", \"FYN\", \"GRB2\", \"CBL\", \"TIGIT\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}