{"gene":"CD226","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1985,"finding":"CD226 (TLiSA1) is a T lineage-specific activation antigen expressed on activated T cells that, when blocked by antibody, inhibits the differentiation of cytotoxic T lymphocytes (CTL) and anomalous killer (AK) cells from their precursors without affecting proliferation, establishing a role in lymphocyte differentiation.","method":"Monoclonal antibody blocking in mixed lymphocyte culture, limiting dilution assay, immunoprecipitation, Scatchard analysis","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 — clean functional blockade with defined phenotypic readout; single lab but multiple orthogonal methods","pmids":["2580933"],"is_preprint":false},{"year":1997,"finding":"CD226 (TLiSA1/PTA1) is a novel member of the immunoglobulin superfamily with two V domains only; identity between the T cell antigen TLiSA1 and platelet PTA1 was established by immunological criteria, internal peptide sequencing from purified platelet glycoprotein, and RT-PCR sequencing of the platelet transcript.","method":"cDNA cloning from Jurkat cells, protein sequencing, RT-PCR, flow cytometry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct molecular characterization with multiple orthogonal methods establishing structure and identity","pmids":["9268302"],"is_preprint":false},{"year":2003,"finding":"CD226 mediates adhesion of thrombin-activated platelets and megakaryocytic cell lines to vascular endothelial cells; thrombin activation induces tyrosine phosphorylation of CD226, and mutation of tyrosine 322 impairs its adhesive function.","method":"Anti-CD226 mAb blocking, mutant transfectant experiments, immunoprecipitation, cell adhesion assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis of active-site residue combined with functional adhesion assays and phosphorylation studies","pmids":["12847109"],"is_preprint":false},{"year":2003,"finding":"CD226 is expressed on activated endothelial cells and mediates adhesion between endothelial cells and leukocytes or tumor cells, as shown by CD226/Ig fusion protein blockade.","method":"Western blot, RT-PCR, CD226/Ig fusion protein blockade, cell adhesion assay","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, functional blockade with fusion protein but limited mechanistic depth","pmids":["12941439"],"is_preprint":false},{"year":2005,"finding":"Serine phosphorylation at residue 329 (S329) of CD226 is required for its recruitment into lipid rafts at the peripheral supra-molecular activation clusters of the immunological synapse; lipid raft association of CD226 is necessary for its tyrosine phosphorylation, which is essential for LFA-1-mediated co-stimulatory signaling for naive T cell proliferation and differentiation.","method":"Mutant transfectants (S329 mutant), lipid raft fractionation, confocal microscopy, T cell proliferation assay","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 1 — site-directed mutagenesis combined with biochemical fractionation and functional readout","pmids":["15684041"],"is_preprint":false},{"year":2005,"finding":"CD226 is specifically expressed on Th1 but not Th2 or Th0 cells; in vivo anti-CD226 treatment reduces Th1 cell expansion, induces APCs that inhibit T cell activation, and delays onset of experimental autoimmune encephalomyelitis, establishing CD226 as a costimulatory molecule for Th1 cells.","method":"Flow cytometry, in vivo antibody treatment, EAE mouse model, APC functional assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — clean in vivo KO/blockade with defined phenotypic readout; single lab","pmids":["16034094"],"is_preprint":false},{"year":2006,"finding":"The CD226 gene contains two promoters (P1 at -810 to -287 bp and P2 at +33 to +213 bp) with a negative regulatory element between them; transcription factor AP-1 upregulates both promoters, and Ets-1 differentially modulates them — promoting AP-1-induced P2 activity while inhibiting AP-1-induced P1 activity via a 10-bp AP-1/Ets-1 composite site.","method":"Promoter-reporter luciferase assays, bioinformatics, transcription factor overexpression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — functional promoter dissection with mutagenesis equivalent; single lab","pmids":["16887814"],"is_preprint":false},{"year":2008,"finding":"DNAM-1 (CD226)-deficient CD8+ T cells require DNAM-1 for co-stimulation when antigen is presented by nonprofessional APCs but not by dendritic cells; DNAM-1-deficient NK cells fail to eliminate tumor cells with few other activating ligands, establishing DNAM-1 as extending the range of target cells that activate lymphocytes.","method":"DNAM-1 knockout mice, in vitro cytotoxicity assays, in vivo tumor challenge","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with multiple in vitro and in vivo orthogonal readouts; replicated by concurrent Iguchi-Manaka paper","pmids":["19029380"],"is_preprint":false},{"year":2008,"finding":"DNAM-1-deficient CTL and NK cells show significantly reduced cytotoxic activity against DNAM-1 ligand-expressing tumors; DNAM-1-deficient mice display accelerated tumor development after transplantation of CD155-expressing tumor cells and after chemical carcinogen treatment, establishing DNAM-1 as required for tumor immunosurveillance in vivo.","method":"DNAM-1 knockout mice, in vitro cytotoxicity assays, MCA/DMBA carcinogen models, tumor transplantation","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with multiple in vivo readouts; replicated by concurrent Gilfillan paper","pmids":["19029379"],"is_preprint":false},{"year":2009,"finding":"CD226 (DNAM-1) on Vγ9Vδ2 T cells mediates cytotoxicity against hepatocellular carcinoma cells expressing Nectin-like-5 (CD155); antibody-mediated masking of DNAM-1 reduces both cytotoxicity and IFN-γ production, and DNAM-1 cooperates with NKG2D in tumor cell lysis.","method":"mAb-mediated blocking, cytotoxicity assays, IFN-γ production assays","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, blocking antibody approach with functional readout","pmids":["19404979"],"is_preprint":false},{"year":2009,"finding":"CD226 is expressed on mouse thymocytes and antagonizes apoptosis; shRNA knockdown of CD226 in fetal thymus organ culture reduces cellularity with enhanced apoptosis, while CD226-transgenic mice show enlarged thymus due to increased cellularity; CD226 regulates survivin expression as part of its anti-apoptotic mechanism.","method":"shRNA knockdown, fetal thymus organ culture, transgenic mice, flow cytometry, Western blot for survivin","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function and gain-of-function approaches with molecular mechanism (survivin); single lab","pmids":["19380793"],"is_preprint":false},{"year":2009,"finding":"Soluble CD226 (sCD226) is shed from cell membranes by proteases (protease inhibitors increase membrane CD226 and decrease sCD226 in supernatants); sCD226-Fc fusion protein dose-dependently inhibits NK cell cytotoxicity against K562 cells.","method":"ELISA, flow cytometry, protease inhibitor treatment, NK cytotoxicity assay with CD226-Fc fusion protein","journal":"BMC immunology","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical shedding mechanism with functional consequence; single lab","pmids":["19490613"],"is_preprint":false},{"year":2012,"finding":"TIGIT exerts immunosuppressive effects on T cells by competing with CD226 for the same CD155 ligand; increases in IFN-γ following TIGIT knockdown could be overcome by blocking CD226 signaling, placing CD226 and TIGIT as competing receptors for CD155 on T cells.","method":"agonistic anti-TIGIT mAb, shRNA TIGIT knockdown, anti-CD226 blocking, IFN-γ/cytokine measurement","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain/loss of function with epistasis; multiple orthogonal methods; replicated across labs","pmids":["22427644"],"is_preprint":false},{"year":2014,"finding":"CD96 competes with CD226 for binding to CD155 and limits NK cell function by direct inhibition; Cd96-/- mice display hyperinflammatory responses to LPS and resistance to carcinogenesis and experimental lung metastases, establishing CD96 as opposing CD226 in NK cell regulation.","method":"Cd96-/- mice, NK cell functional assays, tumor metastasis models, LPS challenge, binding competition assay","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with multiple in vivo and in vitro readouts; highly cited foundational paper","pmids":["24658051"],"is_preprint":false},{"year":2014,"finding":"DNAM-1 is a costimulatory molecule required for optimal expansion and differentiation of MCMV-specific memory NK cells; Src-family kinase Fyn and PKCη signaling through DNAM-1 play distinct roles in generating effector versus memory NK cells during MCMV infection.","method":"Cd226-/- mice, antibody blockade, MCMV infection model, adoptive transfer, signaling inhibitor studies","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO combined with signaling pathway dissection and in vivo memory NK cell differentiation assays","pmids":["24440149"],"is_preprint":false},{"year":2015,"finding":"DNAM-1 (CD226) controls NK cell-mediated cytotoxicity and cytokine production via a conserved tyrosine- and asparagine-based ITT-like motif in its cytoplasmic domain; upon phosphorylation by Src kinases, this motif enables binding to adaptor Grb2, leading to activation of Vav-1, PI3K, PLCγ1, Erk, Akt, and calcium fluxes, as well as increased actin polymerization and granule polarization.","method":"Mutant DNAM-1 transfectants, Co-IP (DNAM-1/Grb2), in vitro kinase assays, cytotoxicity assays, calcium flux measurement, actin polymerization assay, granule polarization imaging","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — reconstitution with mutagenesis and multiple downstream pathway validations; comprehensive mechanistic study","pmids":["26552706"],"is_preprint":false},{"year":2015,"finding":"CD226 expression on Treg cells is associated with reduced suppressive capacity and lineage stability; TIGIT+ Tregs have higher suppressive function, while CD226+TIGIT- Tregs show reduced purity and suppressive capacity after expansion with increased effector cytokine production.","method":"Flow cytometry, in vitro Treg expansion, suppression assays, transcriptional profiling","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — direct cell sorting with functional suppression assays; single lab","pmids":["25994968"],"is_preprint":false},{"year":2015,"finding":"CD226 on donor immune cells (specifically CD4+ T cells and FoxP3+ Tregs) promotes graft-versus-host disease after bone marrow transplantation; absence of DNAM-1 promotes expansion and suppressive function of Treg cells post-BMT while sparing graft-versus-leukemia effects.","method":"DNAM-1-/- donor grafts in MHC-mismatched and MHC-matched BMT models, in vivo Treg expansion assays, GVHD scoring","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with in vivo pathway dissection identifying CD4+ Tregs as the relevant subset","pmids":["23430112"],"is_preprint":false},{"year":2018,"finding":"CD226 regulates NK cell antitumor cytotoxicity through inactivation (phosphorylation) of FOXO1; CD226 engagement by CD155 results in FOXO1 phosphorylation, and FOXO1 phosphorylation inhibitors abrogate CD226-mediated effector responses; CD226 deficiency leads to dysregulated FOXO1-regulated gene expression in tumor-infiltrating NK cells.","method":"Cd226-/- mice, gene expression analysis, in vitro cytotoxicity assays, FOXO1 phosphorylation analysis, FOXO1 inhibitor treatment","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — genetic KO combined with pharmacological inhibition and molecular readouts; multiple orthogonal approaches","pmids":["30504141"],"is_preprint":false},{"year":2018,"finding":"The crystal structure of CD226 ectodomain reveals a unique side-by-side arrangement of two tandem IgV domains (distinct from conventional head-to-tail arrangements); the hybrid complex structure of mouse CD226 ectodomain bound to human CD155-D1 shows that D1 of CD226 forms the conserved binding interface while D2 both stabilizes the architecture and forms direct contacts with CD155; deletion of D2 substantially reduces CD226 binding to CD155.","method":"Crystal structure (X-ray crystallography), surface plasmon resonance binding assay, domain deletion constructs","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation by domain deletion and binding assay","pmids":["30591568"],"is_preprint":false},{"year":2018,"finding":"DNAM-1 is selectively expressed on small peritoneal macrophages (SPMs) but not large peritoneal macrophages (LPMs); DNAM-1 on SPMs mediates CD4+ T cell costimulation during antigen presentation, and DNAM-1 deficiency or blockade impairs CD4+ T cell priming by SPMs.","method":"Flow cytometry, macrophage subset isolation, OVA-specific CD4+ T cell priming assay, DNAM-1 KO mice, antibody blockade","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO combined with blocking antibody and functional co-culture assay; single lab","pmids":["30315271"],"is_preprint":false},{"year":2020,"finding":"Tumor-derived CD155 initiates phosphorylation of CD226 at tyrosine 319 (Y319) by Src kinases, enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation; mutation of Y319 leads to increased CD226 surface expression and enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade.","method":"Y319 mutant CD226, Src kinase inhibitor studies, Co-IP (CD226/CBL-B), ubiquitination assays, in vivo tumor models, flow cytometry","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1 — site-directed mutagenesis combined with biochemical ubiquitination/internalization assays and in vivo functional validation; rigorous mechanistic study","pmids":["33053330"],"is_preprint":false},{"year":2020,"finding":"CD226 loss on CD8+ T cells is driven by an antigen-specific mechanism involving the transcription factor Eomesodermin (Eomes); CD226neg CD8+ T cells exhibit reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program; loss of CD226 impairs response to anti-PD-1 and anti-CD137 immunotherapy.","method":"Cd226-/- mice, Eomes genetic models, transcriptomics, TCR signaling assays, immune checkpoint blockade in vivo","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with mechanistic transcription factor identification and multiple orthogonal functional readouts","pmids":["33053331"],"is_preprint":false},{"year":2020,"finding":"Soluble CD155 (sCD155) secreted by tumor cells preferentially binds DNAM-1 over TIGIT and CD96 and interferes with DNAM-1-mediated NK cell degranulation; in vivo experiments with DNAM-1 KO but not TIGIT KO or CD96 KO mice show loss of sCD155's tumor-promoting effect, establishing DNAM-1 as the primary target of sCD155.","method":"Cd226 KO, TIGIT KO, CD96 KO mice, in vivo tumor colony assays, NK depletion, in vitro degranulation assays, binding affinity comparison","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic KO strains with epistasis and in vitro binding assays; strong mechanistic evidence","pmids":["32040157"],"is_preprint":false},{"year":2020,"finding":"NK cell-derived exosomes carry surface DNAM-1; DNAM-1 on exosomes mediates cytotoxicity against tumor cells, as shown by blocking antibodies to DNAM-1 or its ligands reducing exosome-mediated killing and delaying target cell apoptosis.","method":"Exosome characterization (Western blot, flow cytometry), blocking antibody experiments, cytotoxicity assays","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, blocking antibody approach with functional readout; novel localization finding","pmids":["32178479"],"is_preprint":false},{"year":2021,"finding":"DNAM-1 competes with TIGIT on Treg cells for binding to CD155, thereby regulating TIGIT signaling without delivering its own intracellular signal; DNAM-1 deficiency augments TIGIT signaling, which inhibits the PI3K-mTORC1 pathway, resulting in maintenance of Foxp3 expression and Treg function under inflammatory conditions.","method":"DNAM-1-deficient Treg transfer, GVHD model, Co-IP/competitive binding assays, PI3K-mTORC1 pathway analysis, Foxp3 stability assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with epistasis (DNAM-1 vs TIGIT) and defined intracellular signaling pathway; multiple orthogonal approaches","pmids":["34011606"],"is_preprint":false},{"year":2024,"finding":"CD226 expressed on ILC2s enhances 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 mouse models.","method":"Anti-CD226 antibody blockade, RNA sequencing, flow cytometry, PI3K/AKT and MAPK pathway analysis, Rag2-/- mouse model","journal":"The Journal of allergy and clinical immunology","confidence":"Medium","confidence_rationale":"Tier 2 — functional blockade with signaling pathway identification and in vivo model; single lab","pmids":["38244725"],"is_preprint":false},{"year":2015,"finding":"The MS-associated CD226 genetic variant (rs763361) reduces surface CD226 expression on memory T cells and impairs its induction after stimulation; Foxp3+ Tregs from protective variant carriers show superior suppressive capacity; Cd226-/- mouse Tregs show reduced inhibitory activity with exacerbated EAE, establishing that CD226 regulates Treg function.","method":"Human genetic variant analysis, flow cytometry, Cd226-/- mouse Treg functional assays, EAE model","journal":"Brain","confidence":"Medium","confidence_rationale":"Tier 2 — human genetic variant combined with mouse KO Treg functional assays; single lab but orthogonal approaches","pmids":["26359290"],"is_preprint":false},{"year":2023,"finding":"TIGIT inhibits T cell activation via ligation-induced reorganization into dense nanoclusters at TCR-rich immune synapse clusters, independently of CD226; this inhibitory effect requires TIGIT's intracellular ITT-like signaling motif and reduces cytokine secretion; TIGIT and CD226 co-expression is rare on tumor-infiltrating lymphocytes, indicating CD226-independent TIGIT inhibition is dominant in the tumor microenvironment.","method":"Super-resolution microscopy, flow cytometry of matched tumor/blood samples, TIGIT ITT-like motif mutants, cytokine secretion assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — super-resolution structural imaging combined with mutagenesis and functional assays; multiple orthogonal approaches","pmids":["37596248"],"is_preprint":false}],"current_model":"CD226 (DNAM-1) is a two-IgV-domain immunoglobulin superfamily adhesion and costimulatory receptor that binds CD155 (PVR/Necl-5) and CD112 (nectin-2) via a side-by-side ectodomain arrangement; upon ligand engagement, Src kinases phosphorylate an ITT-like cytoplasmic motif recruiting Grb2 and activating Vav-1/PI3K/PLCγ1/Erk/Akt to drive NK and T cell cytotoxicity, cytokine production, and actin/granule polarization, while tumor-derived CD155 triggers Src-mediated phosphorylation of Y319 enabling CBL-B-dependent ubiquitination and proteasomal degradation of CD226, and CD226 competes with the inhibitory receptor TIGIT for the same ligands to oppose immune suppression, with serine-329 phosphorylation required for lipid raft recruitment and FOXO1 inactivation mediating downstream antitumor responses."},"narrative":{"teleology":[{"year":1985,"claim":"Before molecular identity was known, antibody blockade established that CD226 (TLiSA1) is an activation antigen required for CTL differentiation but not proliferation, defining its functional niche as a costimulatory molecule.","evidence":"Monoclonal antibody blocking in mixed lymphocyte culture with limiting dilution CTL assays","pmids":["2580933"],"confidence":"Medium","gaps":["Molecular identity and ligand unknown","Mechanism of differentiation versus proliferation selectivity unexplained"]},{"year":1997,"claim":"Cloning revealed CD226 as a two-IgV-domain immunoglobulin superfamily member expressed on T cells and platelets, unifying the previously separate TLiSA1 and PTA1 identities and establishing its molecular architecture.","evidence":"cDNA cloning from Jurkat cells, protein sequencing from platelets, RT-PCR","pmids":["9268302"],"confidence":"High","gaps":["Ligand identity unknown","Signaling mechanism unresolved"]},{"year":2003,"claim":"Demonstration that thrombin-induced tyrosine phosphorylation of CD226 (specifically Y322) is required for platelet-endothelial adhesion established CD226 as a phosphorylation-regulated adhesion receptor beyond the lymphocyte lineage.","evidence":"Y322 mutagenesis, immunoprecipitation, platelet/endothelial adhesion assays","pmids":["12847109"],"confidence":"High","gaps":["Kinase responsible for Y322 phosphorylation not identified","Role of CD226 in endothelial contexts only partially explored (PMID:12941439)"]},{"year":2005,"claim":"Identification of S329 phosphorylation as the trigger for lipid raft recruitment and subsequent tyrosine phosphorylation established a two-step activation model linking CD226 membrane partitioning to LFA-1 costimulatory signaling for T cell priming, and selective Th1 expression positioned CD226 in adaptive immune polarization.","evidence":"S329 mutant transfectants, lipid raft fractionation, confocal microscopy, in vivo anti-CD226 in EAE model","pmids":["15684041","16034094"],"confidence":"High","gaps":["Kinase for S329 not identified","Whether Th1 selectivity is transcriptionally controlled was unknown"]},{"year":2008,"claim":"DNAM-1 knockout mice revealed that CD226 is essential for tumor immunosurveillance and extends the range of targets activating NK cells and CD8+ T cells, particularly when other activating ligands are scarce.","evidence":"Cd226−/− mice challenged with CD155-expressing tumors, carcinogen models, in vitro cytotoxicity assays (two independent labs)","pmids":["19029380","19029379"],"confidence":"High","gaps":["Downstream signaling pathway not yet defined at this point","Role of CD226 in memory lymphocyte responses unexplored"]},{"year":2012,"claim":"Epistasis experiments established that TIGIT competes with CD226 for CD155, creating a costimulatory–coinhibitory axis analogous to CD28–CTLA-4, and that CD96 also opposes CD226 in NK cell regulation.","evidence":"TIGIT shRNA knockdown with anti-CD226 blockade, IFN-γ readouts; Cd96−/− mice with tumor and LPS challenge","pmids":["22427644","24658051"],"confidence":"High","gaps":["Relative binding affinities of CD226, TIGIT, and CD96 for CD155 not quantitatively resolved","Cis versus trans competition not distinguished"]},{"year":2014,"claim":"Studies in MCMV infection showed that DNAM-1 is required for memory NK cell differentiation via Fyn and PKCη, extending CD226's role beyond acute effector function to long-lived immunological memory.","evidence":"Cd226−/− mice, MCMV infection model, adoptive transfer, signaling inhibitor studies","pmids":["24440149"],"confidence":"High","gaps":["How Fyn versus PKCη pathways diverge downstream of CD226 incompletely characterized","Whether memory CD8+ T cells similarly depend on CD226 not tested"]},{"year":2015,"claim":"Comprehensive mutagenesis defined the ITT-like motif as the hub for Src-dependent Grb2 recruitment activating Vav-1/PI3K/PLCγ1/Erk/Akt, calcium flux, and actin-driven granule polarization, providing the full intracellular signaling cascade for CD226-mediated cytotoxicity.","evidence":"Mutant DNAM-1 transfectants, Co-IP (DNAM-1/Grb2), in vitro kinase assays, multiple downstream pathway readouts","pmids":["26552706"],"confidence":"High","gaps":["Whether Grb2 is the sole adaptor or others are recruited","Structural basis of the ITT-like motif interaction unresolved"]},{"year":2015,"claim":"CD226 expression on Tregs was shown to reduce suppressive capacity and Foxp3 stability, while an MS-associated CD226 variant (rs763361) altered surface expression and Treg function, linking CD226 to autoimmune disease susceptibility.","evidence":"Treg sorting and suppression assays, human variant analysis, Cd226−/− Treg functional assays, EAE model","pmids":["25994968","26359290"],"confidence":"Medium","gaps":["Mechanism by which CD226 destabilizes Foxp3 not defined at this stage","Whether the rs763361 variant directly alters signaling or only expression levels unclear"]},{"year":2018,"claim":"Structural determination of the CD226 ectodomain revealed its unique side-by-side IgV arrangement and the D1-mediated CD155 binding interface, while functional studies showed CD226 engagement drives FOXO1 phosphorylation as a key effector mechanism for antitumor NK cell responses.","evidence":"X-ray crystallography with domain deletion binding assays; Cd226−/− mice with FOXO1 phosphorylation analysis and inhibitor studies","pmids":["30591568","30504141"],"confidence":"High","gaps":["Full-length CD226–CD155 complex structure at the immune synapse not available","FOXO1 target genes mediating antitumor effect incompletely catalogued"]},{"year":2020,"claim":"Discovery that tumor-derived CD155 triggers Src-dependent Y319 phosphorylation, enabling CBL-B-mediated ubiquitination and proteasomal degradation of CD226, explained how tumors actively downregulate this activating receptor; Eomes-driven transcriptional loss of CD226 on CD8+ T cells was shown to impair checkpoint blockade efficacy.","evidence":"Y319 mutagenesis, CBL-B Co-IP and ubiquitination assays, in vivo tumor models; Eomes genetic models with anti-PD-1/anti-CD137 therapy","pmids":["33053330","33053331"],"confidence":"High","gaps":["Whether CBL-B-dependent degradation operates in all lymphocyte subsets unclear","Therapeutic strategies to block Y319 phosphorylation not developed"]},{"year":2021,"claim":"On Tregs, DNAM-1 was found to regulate TIGIT signaling by competitive ligand sequestration rather than by delivering its own intracellular signal, with DNAM-1 absence augmenting TIGIT-mediated PI3K-mTORC1 inhibition and stabilizing Foxp3 under inflammatory conditions.","evidence":"DNAM-1-deficient Treg transfer in GVHD model, competitive binding and PI3K-mTORC1 pathway analysis","pmids":["34011606"],"confidence":"High","gaps":["Whether this non-signaling competitive mechanism applies to effector T cells","Stoichiometric requirements of CD226–TIGIT competition on single cells unknown"]},{"year":2023,"claim":"Super-resolution imaging revealed that TIGIT can inhibit T cells through nanocluster reorganization independently of CD226, and that TIGIT–CD226 co-expression is rare on tumor-infiltrating lymphocytes, challenging the simple competition model.","evidence":"Super-resolution microscopy, TIGIT ITT-like motif mutants, matched tumor/blood flow cytometry","pmids":["37596248"],"confidence":"High","gaps":["Whether CD226-independent TIGIT inhibition dominates in all tumor types","Spatial relationship between CD226 and TIGIT nanoclusters at the synapse unresolved"]},{"year":2024,"claim":"CD226 function was extended to innate lymphoid cells (ILC2s), where it enhances PI3K/AKT and MAPK signaling and drives airway hyperreactivity, broadening CD226 biology beyond classical lymphocytes.","evidence":"Anti-CD226 blockade, RNA-seq, PI3K/AKT and MAPK analysis in ILC2s, Rag2−/− mouse asthma model","pmids":["38244725"],"confidence":"Medium","gaps":["ILC2-specific signaling partners downstream of CD226 not identified","Whether CD226 on ILC2s competes with TIGIT as it does on NK and T cells untested"]},{"year":null,"claim":"Key unresolved questions include the full-length CD226–CD155 complex structure at the immune synapse, the identity of the S329 kinase, whether therapeutic stabilization of CD226 surface expression (e.g., Y319 mutation) can be exploited clinically, and how CD226 intrinsic signaling versus competitive ligand sequestration is balanced across different lymphocyte subsets.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length synaptic complex structure","S329 kinase identity unknown","In vivo therapeutic targeting of CD226 degradation pathway untested","Cell-type-specific balance of signaling versus competition model unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[2,3,19]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[15,18,26]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12,25]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,4,11,21]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[11,23,24]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7,8,12,14,15,18,22]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,15,18,21,25,26]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,21,22,23]}],"complexes":[],"partners":["PVR","NECTIN2","TIGIT","CD96","GRB2","CBLB","LFA1","FYN"],"other_free_text":[]},"mechanistic_narrative":"CD226 (DNAM-1) is an immunoglobulin superfamily costimulatory receptor that promotes NK cell and T cell cytotoxicity, cytokine production, and tumor immunosurveillance by competing with the inhibitory receptors TIGIT and CD96 for shared ligands CD155 and CD112 [PMID:12847109, PMID:22427644, PMID:24658051, PMID:19029379]. Its crystal structure reveals a unique side-by-side two-IgV-domain ectodomain in which D1 forms the conserved CD155-binding interface and D2 stabilizes the architecture; intracellularly, ligand engagement triggers Src kinase phosphorylation of a conserved ITT-like motif that recruits Grb2 and activates Vav-1/PI3K/PLCγ1/Erk/Akt signaling, driving actin polymerization, granule polarization, and FOXO1 inactivation [PMID:30591568, PMID:26552706, PMID:30504141]. Tumor-derived CD155 exploits CD226 signaling by promoting Src-dependent Y319 phosphorylation, enabling CBL-B-mediated ubiquitination and proteasomal degradation of CD226, which limits antitumor immunity and reduces responsiveness to checkpoint blockade [PMID:33053330, PMID:33053331]. On regulatory T cells, CD226 competes with TIGIT for CD155 binding and thereby modulates TIGIT-dependent PI3K-mTORC1 suppression and Foxp3 stability, positioning the CD226–TIGIT balance as a determinant of Treg function and inflammatory disease outcomes [PMID:34011606, PMID:25994968]."},"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. 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meta-analysis.","date":"2012","source":"Lupus","url":"https://pubmed.ncbi.nlm.nih.gov/22941566","citation_count":19,"is_preprint":false},{"pmid":"33413573","id":"PMC_33413573","title":"Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis.","date":"2021","source":"Arthritis research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33413573","citation_count":19,"is_preprint":false},{"pmid":"21525383","id":"PMC_21525383","title":"DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients.","date":"2011","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/21525383","citation_count":19,"is_preprint":false},{"pmid":"27883251","id":"PMC_27883251","title":"MiR-892a Promotes Hepatocellular Carcinoma Cells Proliferation and Invasion Through Targeting CD226.","date":"2016","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27883251","citation_count":18,"is_preprint":false},{"pmid":"37056782","id":"PMC_37056782","title":"CD226 identifies functional CD8+T cells in the tumor microenvironment and predicts a better outcome for human gastric cancer.","date":"2023","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/37056782","citation_count":18,"is_preprint":false},{"pmid":"31212079","id":"PMC_31212079","title":"Early Cytomegalovirus Reactivation and Expansion of CD56brightCD16dim/-DNAM1+ Natural Killer Cells Are Associated with Antileukemia Effect after Haploidentical Stem Cell Transplantation in Acute Leukemia.","date":"2019","source":"Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation","url":"https://pubmed.ncbi.nlm.nih.gov/31212079","citation_count":17,"is_preprint":false},{"pmid":"35693814","id":"PMC_35693814","title":"Human CD4+CD25+CD226- Tregs Demonstrate Increased Purity, Lineage Stability, and Suppressive Capacity Versus CD4+CD25+CD127lo/- Tregs for Adoptive Cell Therapy.","date":"2022","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35693814","citation_count":17,"is_preprint":false},{"pmid":"25645050","id":"PMC_25645050","title":"Association between the CTLA-4, CD226, FAS polymorphisms and rheumatoid arthritis susceptibility: a meta-analysis.","date":"2015","source":"Human immunology","url":"https://pubmed.ncbi.nlm.nih.gov/25645050","citation_count":17,"is_preprint":false},{"pmid":"31883787","id":"PMC_31883787","title":"Recovery of CD226-TIGIT+FoxP3+ and CD226-TIGIT-FoxP3+ regulatory T cells contributes to clinical remission from active stage in ulcerative colitis patients.","date":"2019","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/31883787","citation_count":16,"is_preprint":false},{"pmid":"21286723","id":"PMC_21286723","title":"The CD226 gene in susceptibility of rheumatoid arthritis in the Chinese Han population.","date":"2011","source":"Rheumatology international","url":"https://pubmed.ncbi.nlm.nih.gov/21286723","citation_count":16,"is_preprint":false},{"pmid":"11152401","id":"PMC_11152401","title":"Preparation and characterization of mabs against different epitopes of CD226 (PTA1).","date":"2000","source":"Hybridoma","url":"https://pubmed.ncbi.nlm.nih.gov/11152401","citation_count":15,"is_preprint":false},{"pmid":"16887814","id":"PMC_16887814","title":"Identification and characterization of the CD226 gene promoter.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16887814","citation_count":15,"is_preprint":false},{"pmid":"2788052","id":"PMC_2788052","title":"Expression of TLiSA1 on T cells from patients with rheumatoid arthritis and systemic lupus erythematosus.","date":"1989","source":"Clinical immunology and immunopathology","url":"https://pubmed.ncbi.nlm.nih.gov/2788052","citation_count":14,"is_preprint":false},{"pmid":"27257974","id":"PMC_27257974","title":"Soluble DNAM-1, as a Predictive Biomarker for Acute Graft-Versus-Host Disease.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27257974","citation_count":14,"is_preprint":false},{"pmid":"19380793","id":"PMC_19380793","title":"Expression of CD226 antagonizes apoptotic cell death in murine thymocytes.","date":"2009","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/19380793","citation_count":14,"is_preprint":false},{"pmid":"36440686","id":"PMC_36440686","title":"NKG2D engagement on human NK cells leads to DNAM-1 hypo-responsiveness through different converging mechanisms.","date":"2022","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/36440686","citation_count":14,"is_preprint":false},{"pmid":"19624611","id":"PMC_19624611","title":"The CD226 gene in susceptibility of type 1 diabetes.","date":"2009","source":"Tissue antigens","url":"https://pubmed.ncbi.nlm.nih.gov/19624611","citation_count":14,"is_preprint":false},{"pmid":"37731380","id":"PMC_37731380","title":"Immune checkpoint molecule DNAM-1/CD112 axis is a novel target for natural killer-cell therapy in acute myeloid leukemia.","date":"2024","source":"Haematologica","url":"https://pubmed.ncbi.nlm.nih.gov/37731380","citation_count":14,"is_preprint":false},{"pmid":"37760586","id":"PMC_37760586","title":"Dysregulation of DNAM-1-Mediated NK Cell Anti-Cancer Responses in the Tumor Microenvironment.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37760586","citation_count":13,"is_preprint":false},{"pmid":"29319370","id":"PMC_29319370","title":"Association between CD226 polymorphism and soluble levels in rheumatoid arthritis: Relationship with clinical activity.","date":"2018","source":"Immunological investigations","url":"https://pubmed.ncbi.nlm.nih.gov/29319370","citation_count":13,"is_preprint":false},{"pmid":"24468679","id":"PMC_24468679","title":"Recombinant soluble CD226 protein directly inhibits cancer cell proliferation in vitro.","date":"2014","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/24468679","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49059,"output_tokens":6713,"usd":0.123936},"stage2":{"model":"claude-opus-4-6","input_tokens":10390,"output_tokens":3654,"usd":0.21495},"total_usd":0.338886,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1985,\n      \"finding\": \"CD226 (TLiSA1) is a T lineage-specific activation antigen expressed on activated T cells that, when blocked by antibody, inhibits the differentiation of cytotoxic T lymphocytes (CTL) and anomalous killer (AK) cells from their precursors without affecting proliferation, establishing a role in lymphocyte differentiation.\",\n      \"method\": \"Monoclonal antibody blocking in mixed lymphocyte culture, limiting dilution assay, immunoprecipitation, Scatchard analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean functional blockade with defined phenotypic readout; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"2580933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CD226 (TLiSA1/PTA1) is a novel member of the immunoglobulin superfamily with two V domains only; identity between the T cell antigen TLiSA1 and platelet PTA1 was established by immunological criteria, internal peptide sequencing from purified platelet glycoprotein, and RT-PCR sequencing of the platelet transcript.\",\n      \"method\": \"cDNA cloning from Jurkat cells, protein sequencing, RT-PCR, flow cytometry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct molecular characterization with multiple orthogonal methods establishing structure and identity\",\n      \"pmids\": [\"9268302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD226 mediates adhesion of thrombin-activated platelets and megakaryocytic cell lines to vascular endothelial cells; thrombin activation induces tyrosine phosphorylation of CD226, and mutation of tyrosine 322 impairs its adhesive function.\",\n      \"method\": \"Anti-CD226 mAb blocking, mutant transfectant experiments, immunoprecipitation, cell adhesion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis of active-site residue combined with functional adhesion assays and phosphorylation studies\",\n      \"pmids\": [\"12847109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD226 is expressed on activated endothelial cells and mediates adhesion between endothelial cells and leukocytes or tumor cells, as shown by CD226/Ig fusion protein blockade.\",\n      \"method\": \"Western blot, RT-PCR, CD226/Ig fusion protein blockade, cell adhesion assay\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, functional blockade with fusion protein but limited mechanistic depth\",\n      \"pmids\": [\"12941439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Serine phosphorylation at residue 329 (S329) of CD226 is required for its recruitment into lipid rafts at the peripheral supra-molecular activation clusters of the immunological synapse; lipid raft association of CD226 is necessary for its tyrosine phosphorylation, which is essential for LFA-1-mediated co-stimulatory signaling for naive T cell proliferation and differentiation.\",\n      \"method\": \"Mutant transfectants (S329 mutant), lipid raft fractionation, confocal microscopy, T cell proliferation assay\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-directed mutagenesis combined with biochemical fractionation and functional readout\",\n      \"pmids\": [\"15684041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD226 is specifically expressed on Th1 but not Th2 or Th0 cells; in vivo anti-CD226 treatment reduces Th1 cell expansion, induces APCs that inhibit T cell activation, and delays onset of experimental autoimmune encephalomyelitis, establishing CD226 as a costimulatory molecule for Th1 cells.\",\n      \"method\": \"Flow cytometry, in vivo antibody treatment, EAE mouse model, APC functional assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean in vivo KO/blockade with defined phenotypic readout; single lab\",\n      \"pmids\": [\"16034094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The CD226 gene contains two promoters (P1 at -810 to -287 bp and P2 at +33 to +213 bp) with a negative regulatory element between them; transcription factor AP-1 upregulates both promoters, and Ets-1 differentially modulates them — promoting AP-1-induced P2 activity while inhibiting AP-1-induced P1 activity via a 10-bp AP-1/Ets-1 composite site.\",\n      \"method\": \"Promoter-reporter luciferase assays, bioinformatics, transcription factor overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional promoter dissection with mutagenesis equivalent; single lab\",\n      \"pmids\": [\"16887814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNAM-1 (CD226)-deficient CD8+ T cells require DNAM-1 for co-stimulation when antigen is presented by nonprofessional APCs but not by dendritic cells; DNAM-1-deficient NK cells fail to eliminate tumor cells with few other activating ligands, establishing DNAM-1 as extending the range of target cells that activate lymphocytes.\",\n      \"method\": \"DNAM-1 knockout mice, in vitro cytotoxicity assays, in vivo tumor challenge\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with multiple in vitro and in vivo orthogonal readouts; replicated by concurrent Iguchi-Manaka paper\",\n      \"pmids\": [\"19029380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNAM-1-deficient CTL and NK cells show significantly reduced cytotoxic activity against DNAM-1 ligand-expressing tumors; DNAM-1-deficient mice display accelerated tumor development after transplantation of CD155-expressing tumor cells and after chemical carcinogen treatment, establishing DNAM-1 as required for tumor immunosurveillance in vivo.\",\n      \"method\": \"DNAM-1 knockout mice, in vitro cytotoxicity assays, MCA/DMBA carcinogen models, tumor transplantation\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with multiple in vivo readouts; replicated by concurrent Gilfillan paper\",\n      \"pmids\": [\"19029379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD226 (DNAM-1) on Vγ9Vδ2 T cells mediates cytotoxicity against hepatocellular carcinoma cells expressing Nectin-like-5 (CD155); antibody-mediated masking of DNAM-1 reduces both cytotoxicity and IFN-γ production, and DNAM-1 cooperates with NKG2D in tumor cell lysis.\",\n      \"method\": \"mAb-mediated blocking, cytotoxicity assays, IFN-γ production assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, blocking antibody approach with functional readout\",\n      \"pmids\": [\"19404979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD226 is expressed on mouse thymocytes and antagonizes apoptosis; shRNA knockdown of CD226 in fetal thymus organ culture reduces cellularity with enhanced apoptosis, while CD226-transgenic mice show enlarged thymus due to increased cellularity; CD226 regulates survivin expression as part of its anti-apoptotic mechanism.\",\n      \"method\": \"shRNA knockdown, fetal thymus organ culture, transgenic mice, flow cytometry, Western blot for survivin\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function and gain-of-function approaches with molecular mechanism (survivin); single lab\",\n      \"pmids\": [\"19380793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Soluble CD226 (sCD226) is shed from cell membranes by proteases (protease inhibitors increase membrane CD226 and decrease sCD226 in supernatants); sCD226-Fc fusion protein dose-dependently inhibits NK cell cytotoxicity against K562 cells.\",\n      \"method\": \"ELISA, flow cytometry, protease inhibitor treatment, NK cytotoxicity assay with CD226-Fc fusion protein\",\n      \"journal\": \"BMC immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical shedding mechanism with functional consequence; single lab\",\n      \"pmids\": [\"19490613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TIGIT exerts immunosuppressive effects on T cells by competing with CD226 for the same CD155 ligand; increases in IFN-γ following TIGIT knockdown could be overcome by blocking CD226 signaling, placing CD226 and TIGIT as competing receptors for CD155 on T cells.\",\n      \"method\": \"agonistic anti-TIGIT mAb, shRNA TIGIT knockdown, anti-CD226 blocking, IFN-γ/cytokine measurement\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss of function with epistasis; multiple orthogonal methods; replicated across labs\",\n      \"pmids\": [\"22427644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD96 competes with CD226 for binding to CD155 and limits NK cell function by direct inhibition; Cd96-/- mice display hyperinflammatory responses to LPS and resistance to carcinogenesis and experimental lung metastases, establishing CD96 as opposing CD226 in NK cell regulation.\",\n      \"method\": \"Cd96-/- mice, NK cell functional assays, tumor metastasis models, LPS challenge, binding competition assay\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with multiple in vivo and in vitro readouts; highly cited foundational paper\",\n      \"pmids\": [\"24658051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DNAM-1 is a costimulatory molecule required for optimal expansion and differentiation of MCMV-specific memory NK cells; Src-family kinase Fyn and PKCη signaling through DNAM-1 play distinct roles in generating effector versus memory NK cells during MCMV infection.\",\n      \"method\": \"Cd226-/- mice, antibody blockade, MCMV infection model, adoptive transfer, signaling inhibitor studies\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO combined with signaling pathway dissection and in vivo memory NK cell differentiation assays\",\n      \"pmids\": [\"24440149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DNAM-1 (CD226) controls NK cell-mediated cytotoxicity and cytokine production via a conserved tyrosine- and asparagine-based ITT-like motif in its cytoplasmic domain; upon phosphorylation by Src kinases, this motif enables binding to adaptor Grb2, leading to activation of Vav-1, PI3K, PLCγ1, Erk, Akt, and calcium fluxes, as well as increased actin polymerization and granule polarization.\",\n      \"method\": \"Mutant DNAM-1 transfectants, Co-IP (DNAM-1/Grb2), in vitro kinase assays, cytotoxicity assays, calcium flux measurement, actin polymerization assay, granule polarization imaging\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with mutagenesis and multiple downstream pathway validations; comprehensive mechanistic study\",\n      \"pmids\": [\"26552706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD226 expression on Treg cells is associated with reduced suppressive capacity and lineage stability; TIGIT+ Tregs have higher suppressive function, while CD226+TIGIT- Tregs show reduced purity and suppressive capacity after expansion with increased effector cytokine production.\",\n      \"method\": \"Flow cytometry, in vitro Treg expansion, suppression assays, transcriptional profiling\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct cell sorting with functional suppression assays; single lab\",\n      \"pmids\": [\"25994968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD226 on donor immune cells (specifically CD4+ T cells and FoxP3+ Tregs) promotes graft-versus-host disease after bone marrow transplantation; absence of DNAM-1 promotes expansion and suppressive function of Treg cells post-BMT while sparing graft-versus-leukemia effects.\",\n      \"method\": \"DNAM-1-/- donor grafts in MHC-mismatched and MHC-matched BMT models, in vivo Treg expansion assays, GVHD scoring\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with in vivo pathway dissection identifying CD4+ Tregs as the relevant subset\",\n      \"pmids\": [\"23430112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD226 regulates NK cell antitumor cytotoxicity through inactivation (phosphorylation) of FOXO1; CD226 engagement by CD155 results in FOXO1 phosphorylation, and FOXO1 phosphorylation inhibitors abrogate CD226-mediated effector responses; CD226 deficiency leads to dysregulated FOXO1-regulated gene expression in tumor-infiltrating NK cells.\",\n      \"method\": \"Cd226-/- mice, gene expression analysis, in vitro cytotoxicity assays, FOXO1 phosphorylation analysis, FOXO1 inhibitor treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO combined with pharmacological inhibition and molecular readouts; multiple orthogonal approaches\",\n      \"pmids\": [\"30504141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The crystal structure of CD226 ectodomain reveals a unique side-by-side arrangement of two tandem IgV domains (distinct from conventional head-to-tail arrangements); the hybrid complex structure of mouse CD226 ectodomain bound to human CD155-D1 shows that D1 of CD226 forms the conserved binding interface while D2 both stabilizes the architecture and forms direct contacts with CD155; deletion of D2 substantially reduces CD226 binding to CD155.\",\n      \"method\": \"Crystal structure (X-ray crystallography), surface plasmon resonance binding assay, domain deletion constructs\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation by domain deletion and binding assay\",\n      \"pmids\": [\"30591568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DNAM-1 is selectively expressed on small peritoneal macrophages (SPMs) but not large peritoneal macrophages (LPMs); DNAM-1 on SPMs mediates CD4+ T cell costimulation during antigen presentation, and DNAM-1 deficiency or blockade impairs CD4+ T cell priming by SPMs.\",\n      \"method\": \"Flow cytometry, macrophage subset isolation, OVA-specific CD4+ T cell priming assay, DNAM-1 KO mice, antibody blockade\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO combined with blocking antibody and functional co-culture assay; single lab\",\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, enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation; mutation of Y319 leads to increased CD226 surface expression and enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade.\",\n      \"method\": \"Y319 mutant CD226, Src kinase inhibitor studies, Co-IP (CD226/CBL-B), ubiquitination assays, in vivo tumor models, flow cytometry\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-directed mutagenesis combined with biochemical ubiquitination/internalization assays and in vivo functional validation; rigorous mechanistic study\",\n      \"pmids\": [\"33053330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD226 loss on CD8+ T cells is driven by an antigen-specific mechanism involving the transcription factor Eomesodermin (Eomes); CD226neg CD8+ T cells exhibit reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program; loss of CD226 impairs response to anti-PD-1 and anti-CD137 immunotherapy.\",\n      \"method\": \"Cd226-/- mice, Eomes genetic models, transcriptomics, TCR signaling assays, immune checkpoint blockade in vivo\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with mechanistic transcription factor identification and multiple orthogonal functional readouts\",\n      \"pmids\": [\"33053331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Soluble CD155 (sCD155) secreted by tumor cells preferentially binds DNAM-1 over TIGIT and CD96 and interferes with DNAM-1-mediated NK cell degranulation; in vivo experiments with DNAM-1 KO but not TIGIT KO or CD96 KO mice show loss of sCD155's tumor-promoting effect, establishing DNAM-1 as the primary target of sCD155.\",\n      \"method\": \"Cd226 KO, TIGIT KO, CD96 KO mice, in vivo tumor colony assays, NK depletion, in vitro degranulation assays, binding affinity comparison\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic KO strains with epistasis and in vitro binding assays; strong mechanistic evidence\",\n      \"pmids\": [\"32040157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NK cell-derived exosomes carry surface DNAM-1; DNAM-1 on exosomes mediates cytotoxicity against tumor cells, as shown by blocking antibodies to DNAM-1 or its ligands reducing exosome-mediated killing and delaying target cell apoptosis.\",\n      \"method\": \"Exosome characterization (Western blot, flow cytometry), blocking antibody experiments, cytotoxicity assays\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, blocking antibody approach with functional readout; novel localization finding\",\n      \"pmids\": [\"32178479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DNAM-1 competes with TIGIT on Treg cells for binding to CD155, thereby regulating TIGIT signaling without delivering its own intracellular signal; DNAM-1 deficiency augments TIGIT signaling, which inhibits the PI3K-mTORC1 pathway, resulting in maintenance of Foxp3 expression and Treg function under inflammatory conditions.\",\n      \"method\": \"DNAM-1-deficient Treg transfer, GVHD model, Co-IP/competitive binding assays, PI3K-mTORC1 pathway analysis, Foxp3 stability assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with epistasis (DNAM-1 vs TIGIT) and defined intracellular signaling pathway; multiple orthogonal approaches\",\n      \"pmids\": [\"34011606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CD226 expressed on ILC2s enhances 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 mouse models.\",\n      \"method\": \"Anti-CD226 antibody blockade, RNA sequencing, flow cytometry, PI3K/AKT and MAPK pathway analysis, Rag2-/- mouse model\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional blockade with signaling pathway identification and in vivo model; single lab\",\n      \"pmids\": [\"38244725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The MS-associated CD226 genetic variant (rs763361) reduces surface CD226 expression on memory T cells and impairs its induction after stimulation; Foxp3+ Tregs from protective variant carriers show superior suppressive capacity; Cd226-/- mouse Tregs show reduced inhibitory activity with exacerbated EAE, establishing that CD226 regulates Treg function.\",\n      \"method\": \"Human genetic variant analysis, flow cytometry, Cd226-/- mouse Treg functional assays, EAE model\",\n      \"journal\": \"Brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — human genetic variant combined with mouse KO Treg functional assays; single lab but orthogonal approaches\",\n      \"pmids\": [\"26359290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TIGIT inhibits T cell activation via ligation-induced reorganization into dense nanoclusters at TCR-rich immune synapse clusters, independently of CD226; this inhibitory effect requires TIGIT's intracellular ITT-like signaling motif and reduces cytokine secretion; TIGIT and CD226 co-expression is rare on tumor-infiltrating lymphocytes, indicating CD226-independent TIGIT inhibition is dominant in the tumor microenvironment.\",\n      \"method\": \"Super-resolution microscopy, flow cytometry of matched tumor/blood samples, TIGIT ITT-like motif mutants, cytokine secretion assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — super-resolution structural imaging combined with mutagenesis and functional assays; multiple orthogonal approaches\",\n      \"pmids\": [\"37596248\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD226 (DNAM-1) is a two-IgV-domain immunoglobulin superfamily adhesion and costimulatory receptor that binds CD155 (PVR/Necl-5) and CD112 (nectin-2) via a side-by-side ectodomain arrangement; upon ligand engagement, Src kinases phosphorylate an ITT-like cytoplasmic motif recruiting Grb2 and activating Vav-1/PI3K/PLCγ1/Erk/Akt to drive NK and T cell cytotoxicity, cytokine production, and actin/granule polarization, while tumor-derived CD155 triggers Src-mediated phosphorylation of Y319 enabling CBL-B-dependent ubiquitination and proteasomal degradation of CD226, and CD226 competes with the inhibitory receptor TIGIT for the same ligands to oppose immune suppression, with serine-329 phosphorylation required for lipid raft recruitment and FOXO1 inactivation mediating downstream antitumor responses.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CD226 (DNAM-1) is an immunoglobulin superfamily costimulatory receptor that promotes NK cell and T cell cytotoxicity, cytokine production, and tumor immunosurveillance by competing with the inhibitory receptors TIGIT and CD96 for shared ligands CD155 and CD112 [PMID:12847109, PMID:22427644, PMID:24658051, PMID:19029379]. Its crystal structure reveals a unique side-by-side two-IgV-domain ectodomain in which D1 forms the conserved CD155-binding interface and D2 stabilizes the architecture; intracellularly, ligand engagement triggers Src kinase phosphorylation of a conserved ITT-like motif that recruits Grb2 and activates Vav-1/PI3K/PLCγ1/Erk/Akt signaling, driving actin polymerization, granule polarization, and FOXO1 inactivation [PMID:30591568, PMID:26552706, PMID:30504141]. Tumor-derived CD155 exploits CD226 signaling by promoting Src-dependent Y319 phosphorylation, enabling CBL-B-mediated ubiquitination and proteasomal degradation of CD226, which limits antitumor immunity and reduces responsiveness to checkpoint blockade [PMID:33053330, PMID:33053331]. On regulatory T cells, CD226 competes with TIGIT for CD155 binding and thereby modulates TIGIT-dependent PI3K-mTORC1 suppression and Foxp3 stability, positioning the CD226–TIGIT balance as a determinant of Treg function and inflammatory disease outcomes [PMID:34011606, PMID:25994968].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Before molecular identity was known, antibody blockade established that CD226 (TLiSA1) is an activation antigen required for CTL differentiation but not proliferation, defining its functional niche as a costimulatory molecule.\",\n      \"evidence\": \"Monoclonal antibody blocking in mixed lymphocyte culture with limiting dilution CTL assays\",\n      \"pmids\": [\"2580933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity and ligand unknown\", \"Mechanism of differentiation versus proliferation selectivity unexplained\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Cloning revealed CD226 as a two-IgV-domain immunoglobulin superfamily member expressed on T cells and platelets, unifying the previously separate TLiSA1 and PTA1 identities and establishing its molecular architecture.\",\n      \"evidence\": \"cDNA cloning from Jurkat cells, protein sequencing from platelets, RT-PCR\",\n      \"pmids\": [\"9268302\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand identity unknown\", \"Signaling mechanism unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstration that thrombin-induced tyrosine phosphorylation of CD226 (specifically Y322) is required for platelet-endothelial adhesion established CD226 as a phosphorylation-regulated adhesion receptor beyond the lymphocyte lineage.\",\n      \"evidence\": \"Y322 mutagenesis, immunoprecipitation, platelet/endothelial adhesion assays\",\n      \"pmids\": [\"12847109\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Y322 phosphorylation not identified\", \"Role of CD226 in endothelial contexts only partially explored (PMID:12941439)\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of S329 phosphorylation as the trigger for lipid raft recruitment and subsequent tyrosine phosphorylation established a two-step activation model linking CD226 membrane partitioning to LFA-1 costimulatory signaling for T cell priming, and selective Th1 expression positioned CD226 in adaptive immune polarization.\",\n      \"evidence\": \"S329 mutant transfectants, lipid raft fractionation, confocal microscopy, in vivo anti-CD226 in EAE model\",\n      \"pmids\": [\"15684041\", \"16034094\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase for S329 not identified\", \"Whether Th1 selectivity is transcriptionally controlled was unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"DNAM-1 knockout mice revealed that CD226 is essential for tumor immunosurveillance and extends the range of targets activating NK cells and CD8+ T cells, particularly when other activating ligands are scarce.\",\n      \"evidence\": \"Cd226−/− mice challenged with CD155-expressing tumors, carcinogen models, in vitro cytotoxicity assays (two independent labs)\",\n      \"pmids\": [\"19029380\", \"19029379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling pathway not yet defined at this point\", \"Role of CD226 in memory lymphocyte responses unexplored\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Epistasis experiments established that TIGIT competes with CD226 for CD155, creating a costimulatory–coinhibitory axis analogous to CD28–CTLA-4, and that CD96 also opposes CD226 in NK cell regulation.\",\n      \"evidence\": \"TIGIT shRNA knockdown with anti-CD226 blockade, IFN-γ readouts; Cd96−/− mice with tumor and LPS challenge\",\n      \"pmids\": [\"22427644\", \"24658051\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative binding affinities of CD226, TIGIT, and CD96 for CD155 not quantitatively resolved\", \"Cis versus trans competition not distinguished\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Studies in MCMV infection showed that DNAM-1 is required for memory NK cell differentiation via Fyn and PKCη, extending CD226's role beyond acute effector function to long-lived immunological memory.\",\n      \"evidence\": \"Cd226−/− mice, MCMV infection model, adoptive transfer, signaling inhibitor studies\",\n      \"pmids\": [\"24440149\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Fyn versus PKCη pathways diverge downstream of CD226 incompletely characterized\", \"Whether memory CD8+ T cells similarly depend on CD226 not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Comprehensive mutagenesis defined the ITT-like motif as the hub for Src-dependent Grb2 recruitment activating Vav-1/PI3K/PLCγ1/Erk/Akt, calcium flux, and actin-driven granule polarization, providing the full intracellular signaling cascade for CD226-mediated cytotoxicity.\",\n      \"evidence\": \"Mutant DNAM-1 transfectants, Co-IP (DNAM-1/Grb2), in vitro kinase assays, multiple downstream pathway readouts\",\n      \"pmids\": [\"26552706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Grb2 is the sole adaptor or others are recruited\", \"Structural basis of the ITT-like motif interaction unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"CD226 expression on Tregs was shown to reduce suppressive capacity and Foxp3 stability, while an MS-associated CD226 variant (rs763361) altered surface expression and Treg function, linking CD226 to autoimmune disease susceptibility.\",\n      \"evidence\": \"Treg sorting and suppression assays, human variant analysis, Cd226−/− Treg functional assays, EAE model\",\n      \"pmids\": [\"25994968\", \"26359290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CD226 destabilizes Foxp3 not defined at this stage\", \"Whether the rs763361 variant directly alters signaling or only expression levels unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Structural determination of the CD226 ectodomain revealed its unique side-by-side IgV arrangement and the D1-mediated CD155 binding interface, while functional studies showed CD226 engagement drives FOXO1 phosphorylation as a key effector mechanism for antitumor NK cell responses.\",\n      \"evidence\": \"X-ray crystallography with domain deletion binding assays; Cd226−/− mice with FOXO1 phosphorylation analysis and inhibitor studies\",\n      \"pmids\": [\"30591568\", \"30504141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length CD226–CD155 complex structure at the immune synapse not available\", \"FOXO1 target genes mediating antitumor effect incompletely catalogued\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that tumor-derived CD155 triggers Src-dependent Y319 phosphorylation, enabling CBL-B-mediated ubiquitination and proteasomal degradation of CD226, explained how tumors actively downregulate this activating receptor; Eomes-driven transcriptional loss of CD226 on CD8+ T cells was shown to impair checkpoint blockade efficacy.\",\n      \"evidence\": \"Y319 mutagenesis, CBL-B Co-IP and ubiquitination assays, in vivo tumor models; Eomes genetic models with anti-PD-1/anti-CD137 therapy\",\n      \"pmids\": [\"33053330\", \"33053331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CBL-B-dependent degradation operates in all lymphocyte subsets unclear\", \"Therapeutic strategies to block Y319 phosphorylation not developed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"On Tregs, DNAM-1 was found to regulate TIGIT signaling by competitive ligand sequestration rather than by delivering its own intracellular signal, with DNAM-1 absence augmenting TIGIT-mediated PI3K-mTORC1 inhibition and stabilizing Foxp3 under inflammatory conditions.\",\n      \"evidence\": \"DNAM-1-deficient Treg transfer in GVHD model, competitive binding and PI3K-mTORC1 pathway analysis\",\n      \"pmids\": [\"34011606\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this non-signaling competitive mechanism applies to effector T cells\", \"Stoichiometric requirements of CD226–TIGIT competition on single cells unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Super-resolution imaging revealed that TIGIT can inhibit T cells through nanocluster reorganization independently of CD226, and that TIGIT–CD226 co-expression is rare on tumor-infiltrating lymphocytes, challenging the simple competition model.\",\n      \"evidence\": \"Super-resolution microscopy, TIGIT ITT-like motif mutants, matched tumor/blood flow cytometry\",\n      \"pmids\": [\"37596248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CD226-independent TIGIT inhibition dominates in all tumor types\", \"Spatial relationship between CD226 and TIGIT nanoclusters at the synapse unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CD226 function was extended to innate lymphoid cells (ILC2s), where it enhances PI3K/AKT and MAPK signaling and drives airway hyperreactivity, broadening CD226 biology beyond classical lymphocytes.\",\n      \"evidence\": \"Anti-CD226 blockade, RNA-seq, PI3K/AKT and MAPK analysis in ILC2s, Rag2−/− mouse asthma model\",\n      \"pmids\": [\"38244725\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ILC2-specific signaling partners downstream of CD226 not identified\", \"Whether CD226 on ILC2s competes with TIGIT as it does on NK and T cells untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full-length CD226–CD155 complex structure at the immune synapse, the identity of the S329 kinase, whether therapeutic stabilization of CD226 surface expression (e.g., Y319 mutation) can be exploited clinically, and how CD226 intrinsic signaling versus competitive ligand sequestration is balanced across different lymphocyte subsets.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length synaptic complex structure\", \"S329 kinase identity unknown\", \"In vivo therapeutic targeting of CD226 degradation pathway untested\", \"Cell-type-specific balance of signaling versus competition model unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [2, 3, 19]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [15, 18, 26]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 4, 11, 21]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [11, 23, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7, 8, 12, 14, 15, 18, 22]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 15, 18, 21, 25, 26]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 21, 22, 23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PVR\",\n      \"NECTIN2\",\n      \"TIGIT\",\n      \"CD96\",\n      \"GRB2\",\n      \"CBLB\",\n      \"LFA1\",\n      \"FYN\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}