{"gene":"NCR3LG1","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":2009,"finding":"B7-H6 (NCR3LG1) is a direct cell-surface ligand for the activating NK cell receptor NKp30, binding to NKp30 and triggering NKp30-mediated NK cell cytotoxicity and cytokine secretion; B7-H6 is not detected on normal tissues but is expressed on tumor cells.","method":"Protein binding assays, functional NK cell activation assays (cytotoxicity and cytokine secretion), flow cytometry, and expression profiling of tumor vs. normal tissues","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1–2 — original discovery with multiple orthogonal methods; replicated extensively across labs","pmids":["19528259"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of NKp30 in complex with B7-H6 revealed that NKp30 engages B7-H6 using both front and back β-sheets of its Ig-like domain (binding via the side and face of the β-sandwich), while B7-H6 contacts NKp30 through the CDR-like loops of its V-like domain in an antibody-like interaction distinct from CTLA-4/B7 and PD-1/PD-L complexes.","method":"X-ray crystallography of NKp30–B7-H6 complex","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with detailed interface characterization","pmids":["21422170"],"is_preprint":false},{"year":2014,"finding":"B7-H6 ectodomain shedding from tumor cell surfaces is mediated by the metalloproteases ADAM10 and ADAM17, generating soluble B7-H6 that impairs NKp30-mediated NK cell activation; pharmacological inhibition or siRNA knockdown of ADAM10/17 increased surface B7-H6 and enhanced NK cell recognition.","method":"Pharmacological inhibitors of ADAM proteases, siRNA-mediated gene attenuation, flow cytometry, NK cell activation assays, ELISA for soluble B7-H6 in patient sera","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (inhibitors + siRNA) in single study, replicated in multiple cancer entities","pmids":["24780758"],"is_preprint":false},{"year":2013,"finding":"B7-H6 expression is induced on CD14+CD16+ proinflammatory monocytes and neutrophils upon stimulation by TLR ligands or proinflammatory cytokines (IL-1β, TNF-α); in these conditions a soluble form is also produced, associated with membrane vesicles in the exosomal fraction.","method":"In vitro stimulation of primary monocytes/neutrophils, flow cytometry for surface B7-H6, ELISA for soluble B7-H6, exosome fractionation/sedimentation","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple methods, in vitro and in vivo (patient samples) evidence","pmids":["23687088"],"is_preprint":false},{"year":2013,"finding":"B7-H6 mRNA and surface protein expression in tumor cells is regulated epigenetically through histone acetylation at the B7-H6 promoter; pan- or class I HDAC inhibitors (or siRNA knockdown of HDAC2/3) downregulate B7-H6, reducing histone acetylation at the promoter and decreasing NKp30-dependent NK effector functions.","method":"HDAC inhibitor treatment, siRNA knockdown of HDAC2/3, luciferase reporter assays, chromatin immunoprecipitation (ChIP), flow cytometry, NK cell functional assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 — ChIP + reporter assays + functional NK readout with multiple orthogonal approaches","pmids":["23801635"],"is_preprint":false},{"year":2016,"finding":"The proto-oncogene c-Myc (and N-Myc) directly drives B7-H6 transcription by binding a functional site in the B7-H6 promoter; pharmacological inhibition or siRNA/shRNA knockdown of c-Myc or N-Myc significantly decreases B7-H6 expression and impairs NKp30-mediated NK cell degranulation.","method":"Luciferase reporter assays, chromatin immunoprecipitation (ChIP), siRNA/shRNA knockdown of c-Myc/N-Myc, pharmacological inhibition, flow cytometry, NK cell degranulation assays","journal":"Oncoimmunology","confidence":"High","confidence_rationale":"Tier 1–2 — ChIP + reporter assay + functional NK readout, multiple tumor cell types","pmids":["27622013"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of antibody 17B1.3 in complex with B7-H6 ectodomain at 2.5 Å resolution showed the antibody binds a site completely distinct from the NKp30-binding site on B7-H6; structure-based mutagenesis of potential B7-H6 dimerization sites did not diminish NKp30-mediated cell activation, indicating the antibody inhibits NK cell activation by sterically interfering with cell–cell contacts rather than directly blocking the NKp30–B7-H6 interaction.","method":"X-ray crystallography at 2.5 Å, structure-based site-directed mutagenesis, NK cell activation assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus functional validation in one study","pmids":["27663271"],"is_preprint":false},{"year":2017,"finding":"Human cytomegalovirus (HCMV) upregulates B7-H6 expression following infection, but HCMV genes US18 and US20 downregulate B7-H6 surface expression via an endosomal degradation mechanism to evade NK cell recognition through NKp30.","method":"HCMV infection of cells, expression of individual viral genes (US18, US20), flow cytometry for surface B7-H6, NK cell killing assays, inhibitors of endosomal/lysosomal degradation","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — viral gene expression with mechanistic follow-up (endosomal degradation pathway) and functional NK readout","pmids":["28819195"],"is_preprint":false},{"year":2015,"finding":"Chronic engagement of NKp30 by surface or soluble B7-H6 on ovarian carcinoma cells leads to downregulation of NKp30 expression on tumor-associated NK cells, impairing their IFN-γ production and cytolytic function.","method":"Flow cytometry of tumor-associated NK cells from peritoneal fluids, functional cytotoxicity and cytokine assays, correlation with surface/soluble B7-H6 levels in peritoneal fluid","journal":"Oncoimmunology","confidence":"Medium","confidence_rationale":"Tier 2 — patient sample phenotyping with functional assays; single cohort study","pmids":["26137398"],"is_preprint":false},{"year":2015,"finding":"Soluble B7-H6 in serum of high-risk neuroblastoma patients inhibits NK cell functions in vitro, correlating with NKp30 downregulation; NB cells expressing surface B7-H6 stimulate NK cells in an NKp30-dependent manner.","method":"In vitro NK cell functional assays with patient serum, NKp30-blocking antibodies, flow cytometry, ELISA for soluble B7-H6","journal":"Science translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 — receptor-blocking antibody experiments plus patient serum functional inhibition, single lab","pmids":["25877893"],"is_preprint":false},{"year":2015,"finding":"Tumor therapeutic stressors (cisplatin, 5-fluorouracil, radiation, heat shock, TNF-α) upregulate B7-H6 surface expression on tumor cells, enhancing their susceptibility to NKp30-mediated NK cell lysis; B7-H6 shRNA knockdown dampened this sensitization.","method":"Drug/radiation/heat-shock treatment of tumor cell lines, flow cytometry for B7-H6, NK cell cytotoxicity assays, shRNA knockdown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple stressors tested, shRNA rescue experiment, functional NK readout","pmids":["26472927"],"is_preprint":false},{"year":2021,"finding":"Soluble B7-H6 (sB7-H6) inhibits NK cell-mediated target cell killing and is associated with diminished cell surface expression of NKG2D and NCRs (NKp30, NKp46); in contrast to BAG6, B7-H6 is only marginally released in association with extracellular vesicles and EV-associated B7-H6 does not stimulate NK killing.","method":"Recombinant ligand production in HEK293 cells, NK cell killing assays, flow cytometry for receptor expression, nano flow cytometry of EVs, EV isolation and characterization","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods in one study comparing soluble vs. EV-associated forms","pmids":["33671836"],"is_preprint":false},{"year":2021,"finding":"BRD4, an epigenetic reader, activates B7-H6 transcription in AML cells: histone acetylation mediated by CBP30/P300 facilitates BRD4 binding to the B7-H6 promoter, which recruits P-TEFb to phosphorylate RNA polymerase II; BRD4 also co-binds with JMJD6 at a distal B7-H6 enhancer. Metformin reduces histone acetylation at the B7-H6 promoter, impairing BRD4 binding and inhibiting B7-H6 expression.","method":"BRD4-specific inhibition, ChIP for BRD4/histone acetylation/P-TEFb at B7-H6 promoter/enhancer, flow cytometry, RT-PCR, metformin treatment, B7-H6 knockdown apoptosis assays","journal":"Oncoimmunology","confidence":"High","confidence_rationale":"Tier 1–2 — ChIP with multiple marks plus functional inhibition experiments in a single rigorous study","pmids":["33796404"],"is_preprint":false},{"year":2023,"finding":"B7-H6 contains six functional N-linked glycosylation sites; N43 and N208 glycosylation are conserved in jawed vertebrates and are essential for NK cell activation. Mechanistically, N43 glycosylation contributes to B7-H6/NKp30 binding affinity, while N208 glycosylation is required for membrane stability (lack of N208 leads to B7-H6 shedding from the membrane).","method":"PNGase F digestion, tunicamycin blockage, single-site mutagenesis, SDS-PAGE/Western blot, flow cytometry, NK cell activation assays, phylogenetic and structural analysis","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 1 — systematic site mutagenesis with in vitro functional validation and mechanistic dissection of individual glycan roles","pmids":["38035117"],"is_preprint":false},{"year":2019,"finding":"B7-H6 is strongly expressed on the surface of CD4+ T cells infected with HIV-2, and chronic NKp30 engagement by this surface B7-H6 leads to downregulation of NKp30 on NK cells, impairing their cytolytic function while increasing IFN-γ overproduction.","method":"Flow cytometry phenotyping of NK cells and target cells from HIV-2 patients, functional killing assays against B7-H6-expressing targets","journal":"AIDS","confidence":"Medium","confidence_rationale":"Tier 2 — patient cohort with functional assays; correlative but mechanistically linked to B7-H6 surface expression on infected cells","pmids":["30325780"],"is_preprint":false},{"year":2020,"finding":"B7-H6 promotes tumor cell proliferation, migration, and invasion in non-Hodgkin lymphoma via the Ras/MEK/ERK signaling pathway; B7-H6 knockdown inhibited Ras/MEK/ERK phosphorylation, and MEK inhibitor AZD8330 reversed migration/invasion induced by B7-H6.","method":"B7-H6 knockdown and overexpression in NHL cell lines, quantitative phosphoproteomics, Western blot for Ras/MEK/ERK pathway, MEK inhibitor rescue, in vivo xenograft model, CCK-8 and Transwell assays","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 — phosphoproteomics plus pathway inhibitor rescue plus in vivo validation","pmids":["32606790"],"is_preprint":false},{"year":2020,"finding":"B7-H6 preferentially expressed in glioma stem-like cells (GSLCs) promotes proliferation via the c-Myc/RNMT axis; B7-H6 knockdown decreased c-Myc expression, inhibited PI3K/Akt and ERK/MAPK signaling, and specifically reduced RNMT (RNA guanine-7 methyltransferase) expression among co-expressed genes. CRISPR-Cas9 knockout of B7-H6 in 293T cells also suppressed proliferation.","method":"siRNA knockdown, CRISPR-Cas9 knockout, Western blot for c-Myc/PI3K/Akt/ERK, TCGA co-expression analysis, flow cytometry, CCK-8 proliferation assays","journal":"Journal of immunology research","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO plus siRNA plus signaling pathway analysis; single lab","pmids":["32322592"],"is_preprint":false},{"year":2021,"finding":"B7-H6 undergoes nuclear translocation in T-lymphoblastic lymphoma (T-LBL) cells, with a specific nuclear localization sequence identified in the C-terminus; B7-H6 depletion in Jurkat cells impaired proliferation, migration, and invasion, and RNA-seq revealed differential expression of RAG-1 as a potential downstream mediator.","method":"Immunohistochemistry showing nuclear localization, nuclear localization sequence analysis, B7-H6 depletion, RNA sequencing, functional cell assays","journal":"Annals of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — localization finding with functional KD and RNAseq; single lab","pmids":["33708955"],"is_preprint":false},{"year":2025,"finding":"SNRPA promotes B7-H6 expression in hepatocellular carcinoma by directly binding B7-H6 pre-mRNA and facilitating its maturation through intron 2 splicing; SNRPA-driven B7-H6 upregulation activates the B7-H6–STAT3/AKT signaling axis, promoting G1-S transition and inhibiting apoptosis, and confers resistance to lenvatinib.","method":"Nanopore full-length cDNA sequencing, RNA-binding protein immunoprecipitation sequencing, RIP-PCR with exon-exon and exon-intron junction primers, gain/loss-of-function experiments, Western blot for STAT3/AKT, flow cytometry, drug sensitivity assays","journal":"Bioscience trends","confidence":"Medium","confidence_rationale":"Tier 2 — RNA-binding/splicing mechanism with multiple orthogonal methods; single lab","pmids":["40240166"],"is_preprint":false},{"year":2021,"finding":"B7-H6 knockdown in AML (HEL-R) cells induces apoptosis, and high B7-H6 expression in AML activates anti-apoptotic signaling; the BRD4–B7-H6 regulatory axis is linked to the STAT3 pathway activity in these cells.","method":"B7-H6 knockdown (siRNA), flow cytometry for apoptosis, Western blot, correlation with BRD4 inhibition","journal":"Oncoimmunology","confidence":"Medium","confidence_rationale":"Tier 2 — functional KD with apoptosis readout in same study as ChIP mechanistic work","pmids":["33796404"],"is_preprint":false},{"year":2015,"finding":"NKp30-B7-H6 interaction in HBV-related acute-on-chronic liver failure leads to upregulation of IL-32 in NK cells; IL-32 in turn induces apoptosis of hepatocytes in a dose-dependent manner, establishing a mechanistic link between B7-H6 engagement and hepatocyte damage via IL-32.","method":"Co-culture of NK-92 cells with B7-H6-expressing hepatocytes, anti-NKp30 antibody stimulation, flow cytometry for IL-32 in NK cells, Annexin V/PI apoptosis assay of hepatocytes treated with IL-32, immunohistochemistry in patient liver samples","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro functional assays with receptor-blocking antibody and recombinant IL-32 dose-response; single lab","pmids":["26241657"],"is_preprint":false},{"year":2021,"finding":"atRA-induced downregulation of c-Myc reduces B7-H6 transcription in leukemia cells, rendering them resistant to NKp30/B7-H6-dependent NK cell killing; epigenetic modifications (DNA methylation, HDAC) were not responsible, as their inhibitors could not restore B7-H6 expression.","method":"CRISPR/Cas9 B7-H6 knockdown, siRNA c-Myc knockdown, atRA treatment, real-time PCR, flow cytometry, Western blot, NK cell cytotoxicity assays, HDAC/DNA methylation inhibitor controls","journal":"Cancer communications","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO plus siRNA plus pharmacological controls, functional NK readout; single lab","pmids":["34236140"],"is_preprint":false},{"year":2024,"finding":"CRISPR-mediated knockout of B7-H6 in CD4+ T cells reduced NK cell responses to HIV-1-infected cells, confirming the NKp30:B7-H6 receptor–ligand pair as functionally required for NK recognition of HIV-infected cells.","method":"CRISPR editing of CD4+ T cells to knockout B7-H6, NK cell functional assays, NKp30 overexpression in NK cells","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO with functional NK readout; preprint, single lab","pmids":["bio_10.1101_2024.06.24.600449"],"is_preprint":true},{"year":2023,"finding":"B7-H6 promotes medulloblastoma cell migration and invasion by activating c-MYC expression, which in turn drives F-actin cytoskeletal rearrangement and upregulates MMP-2 and MMP-9.","method":"siRNA knockdown and plasmid overexpression of B7-H6, Transwell migration/invasion assays, Western blot for c-MYC/MMP-2/MMP-9, immunofluorescence staining for F-actin","journal":"Medical oncology","confidence":"Low","confidence_rationale":"Tier 3 — single lab, KD/OE with downstream pathway markers; no reconstitution or ChIP","pmids":["36692844"],"is_preprint":false},{"year":2025,"finding":"G-CSF derived from gastric cancer tumors induces B7-H6 expression on neutrophils via NF-κB signaling pathway activation; B7-H6 on tumor-infiltrating neutrophils promotes their survival by blocking apoptosis, as B7-H6 blockade accelerated neutrophil apoptosis and shortened their lifespan.","method":"Flow cytometry phenotyping, in vitro neutrophil stimulation with G-CSF and tumor-conditioned supernatants, NF-κB pathway analysis, B7-H6 blockade with antibodies, apoptosis assays","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (stimulation + pathway inhibition + receptor blockade) with functional apoptosis readout","pmids":["40054066"],"is_preprint":false}],"current_model":"NCR3LG1/B7-H6 is a tumor- and stress-induced cell-surface ligand belonging to the B7 family that binds directly to the activating NK cell receptor NKp30 (as defined by crystal structure of the complex), triggering NK cell cytotoxicity and cytokine secretion; its surface expression is transcriptionally driven by c-Myc and epigenetically regulated via BRD4/histone acetylation and HDAC activity at its promoter, post-translationally controlled by N-glycosylation at N43 (NKp30 binding) and N208 (membrane stability), and proteolytically shed from the cell surface by ADAM10/ADAM17 metalloproteases to generate immunosuppressive soluble B7-H6 that downregulates NKp30 on NK cells; intracellularly, B7-H6 promotes tumor cell-autonomous oncogenic signaling through c-Myc, Ras/MEK/ERK, PI3K/Akt, and STAT3 pathways to support proliferation, migration, invasion, and survival."},"narrative":{"teleology":[{"year":2009,"claim":"Identification of B7-H6 as the long-sought cellular ligand for the orphan activating NK receptor NKp30 established the molecular basis for NKp30-dependent tumor immunosurveillance.","evidence":"Protein binding assays, functional NK cytotoxicity/cytokine assays, flow cytometry, and expression profiling across tumor vs. normal tissues","pmids":["19528259"],"confidence":"High","gaps":["Structural basis of interaction unknown","Regulation of B7-H6 expression not addressed","Whether soluble forms exist and their function unclear"]},{"year":2011,"claim":"Crystal structure of the NKp30–B7-H6 complex revealed an unconventional binding mode distinct from other B7-family immune checkpoint interactions, explaining NKp30's unique recognition strategy.","evidence":"X-ray crystallography of the NKp30–B7-H6 complex","pmids":["21422170"],"confidence":"High","gaps":["Role of glycosylation in binding not resolved","Whether B7-H6 dimerization is functionally relevant unclear","Mechanism of signal transduction downstream of NKp30 engagement not addressed"]},{"year":2013,"claim":"B7-H6 expression was shown to extend beyond tumors to activated inflammatory myeloid cells, and its transcription was found to be epigenetically controlled through histone acetylation at its promoter, broadening understanding of B7-H6 regulation and its role in non-tumor immunity.","evidence":"In vitro stimulation of monocytes/neutrophils with TLR ligands and cytokines; HDAC inhibitor treatment, siRNA knockdown of HDAC2/3, ChIP for histone acetylation at B7-H6 promoter, luciferase reporters, functional NK assays","pmids":["23687088","23801635"],"confidence":"High","gaps":["Specific transcription factors driving B7-H6 promoter activity not identified","In vivo relevance of myeloid B7-H6 in infection/inflammation not established","Whether exosomal B7-H6 is functionally active on NK cells unclear"]},{"year":2014,"claim":"Discovery that ADAM10/ADAM17 metalloproteases shed B7-H6 ectodomain from tumor cells established that soluble B7-H6 functions as an immunosuppressive decoy impairing NKp30-mediated NK activation.","evidence":"Pharmacological ADAM inhibitors, siRNA knockdown of ADAM10/17, flow cytometry, NK activation assays, ELISA for soluble B7-H6 in patient sera","pmids":["24780758"],"confidence":"High","gaps":["Cleavage site on B7-H6 not mapped","Relative contributions of ADAM10 vs. ADAM17 in different tumor types not resolved","Whether shedding is regulated by upstream signals unknown"]},{"year":2015,"claim":"Multiple studies demonstrated that therapeutic stress upregulates surface B7-H6 to sensitize tumor cells to NK lysis, while chronic B7-H6 exposure (surface or soluble) in ovarian carcinoma and neuroblastoma downregulates NKp30 to exhaust NK cells, revealing B7-H6's dual role as both immune activator and suppressor depending on context.","evidence":"Drug/radiation/heat-shock treatment of tumor lines with shRNA rescue; patient tumor-associated NK cell phenotyping from peritoneal fluids; patient serum functional inhibition assays with NKp30-blocking antibodies","pmids":["26472927","26137398","25877893"],"confidence":"Medium","gaps":["Signaling pathways governing stress-induced B7-H6 upregulation not identified","Threshold of B7-H6 exposure triggering NKp30 downregulation not quantified","Whether NKp30 downregulation is reversible in vivo not tested"]},{"year":2016,"claim":"Identification of c-Myc/N-Myc as direct transcriptional activators of B7-H6 provided the first specific transcription factor–promoter mechanism and linked oncogene-driven expression to NK immune recognition.","evidence":"ChIP showing c-Myc binding at B7-H6 promoter, luciferase reporter assays, siRNA/shRNA knockdown of c-Myc/N-Myc, NK degranulation assays across multiple tumor types","pmids":["27622013"],"confidence":"High","gaps":["Whether other oncogenic transcription factors contribute to B7-H6 expression not addressed","Post-transcriptional regulation not explored","Whether c-Myc also drives B7-H6 in non-tumor cells unknown"]},{"year":2017,"claim":"HCMV proteins US18 and US20 were shown to downregulate B7-H6 via endosomal degradation, establishing a viral immune evasion mechanism targeting the NKp30–B7-H6 axis.","evidence":"Expression of individual HCMV genes in cells, flow cytometry, NK killing assays, endosomal/lysosomal degradation inhibitors","pmids":["28819195"],"confidence":"High","gaps":["Whether other herpesviruses target B7-H6 not tested","Specific endosomal trafficking machinery involved not identified","Whether US18/US20 directly bind B7-H6 not shown"]},{"year":2020,"claim":"B7-H6 was found to have tumor cell-autonomous signaling functions, promoting proliferation, migration, and invasion through Ras/MEK/ERK and PI3K/Akt/c-Myc pathways independent of its NK ligand role.","evidence":"B7-H6 knockdown/overexpression in NHL and glioma cell lines, phosphoproteomics, MEK inhibitor rescue, Western blot for pathway components, xenograft models, CRISPR-Cas9 KO in 293T cells","pmids":["32606790","32322592"],"confidence":"Medium","gaps":["Whether B7-H6 signals through a cis-receptor or acts receptor-independently is unknown","No binding partner on the tumor cell surface identified for intrinsic signaling","Single-lab findings for each tumor type"]},{"year":2021,"claim":"BRD4 was identified as an epigenetic reader that activates B7-H6 transcription by recognizing acetylated histones at the promoter and recruiting P-TEFb; additionally, atRA-mediated c-Myc downregulation was shown to reduce B7-H6 and confer NK resistance, unifying transcriptional and epigenetic control.","evidence":"ChIP for BRD4, histone acetylation, and P-TEFb at B7-H6 promoter/enhancer; BRD4 inhibition; metformin treatment; atRA treatment with CRISPR/siRNA controls and NK cytotoxicity assays","pmids":["33796404","34236140"],"confidence":"High","gaps":["Whether BRD4 and c-Myc cooperate at the B7-H6 promoter or act independently not resolved","Enhancer–promoter looping mechanism not demonstrated","In vivo therapeutic validation of BRD4/c-Myc targeting to modulate B7-H6 lacking"]},{"year":2023,"claim":"Systematic glycosylation mapping revealed that conserved N-linked glycans at N43 and N208 serve distinct mechanistic roles—N43 for NKp30 binding affinity and N208 for membrane retention—explaining how post-translational modification fine-tunes both immune recognition and shedding.","evidence":"PNGase F digestion, tunicamycin treatment, single-site mutagenesis of six N-glycosylation sites, flow cytometry, NK activation assays, phylogenetic analysis","pmids":["38035117"],"confidence":"High","gaps":["Glycan composition at each site not determined","Whether glycosylation status changes under stress or in different tumor types unknown","How N208 glycosylation mechanistically prevents shedding not resolved"]},{"year":2025,"claim":"SNRPA was identified as an RNA-binding protein that promotes B7-H6 pre-mRNA maturation via intron 2 splicing, connecting RNA processing to B7-H6-driven STAT3/AKT signaling and lenvatinib resistance in hepatocellular carcinoma; separately, G-CSF-induced NF-κB signaling was shown to drive B7-H6 on tumor-infiltrating neutrophils to promote their survival.","evidence":"Nanopore sequencing, RIP-seq/RIP-PCR with exon-junction primers, gain/loss-of-function for SNRPA, Western blot; G-CSF stimulation, NF-κB pathway analysis, B7-H6 antibody blockade, apoptosis assays","pmids":["40240166","40054066"],"confidence":"Medium","gaps":["SNRPA–B7-H6 splicing mechanism needs independent validation","Whether SNRPA regulation of B7-H6 extends beyond HCC not tested","How B7-H6 blocks neutrophil apoptosis mechanistically (cis-signaling vs. receptor engagement) remains undefined"]},{"year":null,"claim":"The mechanism by which B7-H6 transduces cell-autonomous oncogenic signals (Ras/MEK/ERK, PI3K/Akt, STAT3) remains unknown—no cis-receptor, intracellular binding partner, or signaling domain has been identified to explain tumor-intrinsic B7-H6 function.","evidence":"","pmids":[],"confidence":"Low","gaps":["No intracellular binding partners identified","No signaling motif in B7-H6 cytoplasmic tail characterized","Relationship between nuclear-localized B7-H6 and transcriptional effects unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[15,16,18,19]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,3,7,13]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[2,8,9,11]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,2,8,9,10,11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[15,16,18,24]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[15,16,18,19]}],"complexes":[],"partners":["NCR3","ADAM10","ADAM17","MYC","BRD4","SNRPA"],"other_free_text":[]},"mechanistic_narrative":"NCR3LG1 (B7-H6) is a B7-family cell-surface ligand that bridges innate immune surveillance and tumor-intrinsic oncogenic signaling. B7-H6 binds directly to the activating NK cell receptor NKp30 through CDR-like loops of its V-like domain, triggering NK cell cytotoxicity and cytokine secretion; this interaction is structurally distinct from canonical B7/CTLA-4 or PD-1/PD-L engagement [PMID:19528259, PMID:21422170]. Surface expression is transcriptionally driven by c-Myc binding at the B7-H6 promoter and epigenetically regulated via BRD4-mediated histone acetylation, while post-translational N-glycosylation at N43 tunes NKp30 binding affinity and at N208 controls membrane stability; ADAM10/ADAM17-mediated ectodomain shedding generates immunosuppressive soluble B7-H6 that downregulates NKp30 on NK cells [PMID:27622013, PMID:33796404, PMID:38035117, PMID:24780758]. Beyond its immune-ligand role, B7-H6 promotes tumor cell proliferation, migration, and survival through cell-autonomous activation of Ras/MEK/ERK, PI3K/Akt, and STAT3 pathways, and its expression on inflammatory monocytes, neutrophils, and virally infected cells extends its function to non-tumor immune contexts [PMID:32606790, PMID:32322592, PMID:23687088, PMID:40054066]."},"prefetch_data":{"uniprot":{"accession":"Q68D85","full_name":"Natural cytotoxicity triggering receptor 3 ligand 1","aliases":["B7 homolog 6","B7-H6"],"length_aa":454,"mass_kda":50.8,"function":"Triggers NCR3-dependent natural killer cell activation","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q68D85/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NCR3LG1","classification":"Not 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B7-H6 is not detected on normal tissues but is expressed on tumor cells.\",\n      \"method\": \"Protein binding assays, functional NK cell activation assays (cytotoxicity and cytokine secretion), flow cytometry, and expression profiling of tumor vs. normal tissues\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — original discovery with multiple orthogonal methods; replicated extensively across labs\",\n      \"pmids\": [\"19528259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of NKp30 in complex with B7-H6 revealed that NKp30 engages B7-H6 using both front and back β-sheets of its Ig-like domain (binding via the side and face of the β-sandwich), while B7-H6 contacts NKp30 through the CDR-like loops of its V-like domain in an antibody-like interaction distinct from CTLA-4/B7 and PD-1/PD-L complexes.\",\n      \"method\": \"X-ray crystallography of NKp30–B7-H6 complex\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with detailed interface characterization\",\n      \"pmids\": [\"21422170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"B7-H6 ectodomain shedding from tumor cell surfaces is mediated by the metalloproteases ADAM10 and ADAM17, generating soluble B7-H6 that impairs NKp30-mediated NK cell activation; pharmacological inhibition or siRNA knockdown of ADAM10/17 increased surface B7-H6 and enhanced NK cell recognition.\",\n      \"method\": \"Pharmacological inhibitors of ADAM proteases, siRNA-mediated gene attenuation, flow cytometry, NK cell activation assays, ELISA for soluble B7-H6 in patient sera\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (inhibitors + siRNA) in single study, replicated in multiple cancer entities\",\n      \"pmids\": [\"24780758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"B7-H6 expression is induced on CD14+CD16+ proinflammatory monocytes and neutrophils upon stimulation by TLR ligands or proinflammatory cytokines (IL-1β, TNF-α); in these conditions a soluble form is also produced, associated with membrane vesicles in the exosomal fraction.\",\n      \"method\": \"In vitro stimulation of primary monocytes/neutrophils, flow cytometry for surface B7-H6, ELISA for soluble B7-H6, exosome fractionation/sedimentation\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods, in vitro and in vivo (patient samples) evidence\",\n      \"pmids\": [\"23687088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"B7-H6 mRNA and surface protein expression in tumor cells is regulated epigenetically through histone acetylation at the B7-H6 promoter; pan- or class I HDAC inhibitors (or siRNA knockdown of HDAC2/3) downregulate B7-H6, reducing histone acetylation at the promoter and decreasing NKp30-dependent NK effector functions.\",\n      \"method\": \"HDAC inhibitor treatment, siRNA knockdown of HDAC2/3, luciferase reporter assays, chromatin immunoprecipitation (ChIP), flow cytometry, NK cell functional assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP + reporter assays + functional NK readout with multiple orthogonal approaches\",\n      \"pmids\": [\"23801635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The proto-oncogene c-Myc (and N-Myc) directly drives B7-H6 transcription by binding a functional site in the B7-H6 promoter; pharmacological inhibition or siRNA/shRNA knockdown of c-Myc or N-Myc significantly decreases B7-H6 expression and impairs NKp30-mediated NK cell degranulation.\",\n      \"method\": \"Luciferase reporter assays, chromatin immunoprecipitation (ChIP), siRNA/shRNA knockdown of c-Myc/N-Myc, pharmacological inhibition, flow cytometry, NK cell degranulation assays\",\n      \"journal\": \"Oncoimmunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP + reporter assay + functional NK readout, multiple tumor cell types\",\n      \"pmids\": [\"27622013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of antibody 17B1.3 in complex with B7-H6 ectodomain at 2.5 Å resolution showed the antibody binds a site completely distinct from the NKp30-binding site on B7-H6; structure-based mutagenesis of potential B7-H6 dimerization sites did not diminish NKp30-mediated cell activation, indicating the antibody inhibits NK cell activation by sterically interfering with cell–cell contacts rather than directly blocking the NKp30–B7-H6 interaction.\",\n      \"method\": \"X-ray crystallography at 2.5 Å, structure-based site-directed mutagenesis, NK cell activation assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus functional validation in one study\",\n      \"pmids\": [\"27663271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human cytomegalovirus (HCMV) upregulates B7-H6 expression following infection, but HCMV genes US18 and US20 downregulate B7-H6 surface expression via an endosomal degradation mechanism to evade NK cell recognition through NKp30.\",\n      \"method\": \"HCMV infection of cells, expression of individual viral genes (US18, US20), flow cytometry for surface B7-H6, NK cell killing assays, inhibitors of endosomal/lysosomal degradation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — viral gene expression with mechanistic follow-up (endosomal degradation pathway) and functional NK readout\",\n      \"pmids\": [\"28819195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Chronic engagement of NKp30 by surface or soluble B7-H6 on ovarian carcinoma cells leads to downregulation of NKp30 expression on tumor-associated NK cells, impairing their IFN-γ production and cytolytic function.\",\n      \"method\": \"Flow cytometry of tumor-associated NK cells from peritoneal fluids, functional cytotoxicity and cytokine assays, correlation with surface/soluble B7-H6 levels in peritoneal fluid\",\n      \"journal\": \"Oncoimmunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — patient sample phenotyping with functional assays; single cohort study\",\n      \"pmids\": [\"26137398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Soluble B7-H6 in serum of high-risk neuroblastoma patients inhibits NK cell functions in vitro, correlating with NKp30 downregulation; NB cells expressing surface B7-H6 stimulate NK cells in an NKp30-dependent manner.\",\n      \"method\": \"In vitro NK cell functional assays with patient serum, NKp30-blocking antibodies, flow cytometry, ELISA for soluble B7-H6\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — receptor-blocking antibody experiments plus patient serum functional inhibition, single lab\",\n      \"pmids\": [\"25877893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Tumor therapeutic stressors (cisplatin, 5-fluorouracil, radiation, heat shock, TNF-α) upregulate B7-H6 surface expression on tumor cells, enhancing their susceptibility to NKp30-mediated NK cell lysis; B7-H6 shRNA knockdown dampened this sensitization.\",\n      \"method\": \"Drug/radiation/heat-shock treatment of tumor cell lines, flow cytometry for B7-H6, NK cell cytotoxicity assays, shRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple stressors tested, shRNA rescue experiment, functional NK readout\",\n      \"pmids\": [\"26472927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Soluble B7-H6 (sB7-H6) inhibits NK cell-mediated target cell killing and is associated with diminished cell surface expression of NKG2D and NCRs (NKp30, NKp46); in contrast to BAG6, B7-H6 is only marginally released in association with extracellular vesicles and EV-associated B7-H6 does not stimulate NK killing.\",\n      \"method\": \"Recombinant ligand production in HEK293 cells, NK cell killing assays, flow cytometry for receptor expression, nano flow cytometry of EVs, EV isolation and characterization\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in one study comparing soluble vs. EV-associated forms\",\n      \"pmids\": [\"33671836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BRD4, an epigenetic reader, activates B7-H6 transcription in AML cells: histone acetylation mediated by CBP30/P300 facilitates BRD4 binding to the B7-H6 promoter, which recruits P-TEFb to phosphorylate RNA polymerase II; BRD4 also co-binds with JMJD6 at a distal B7-H6 enhancer. Metformin reduces histone acetylation at the B7-H6 promoter, impairing BRD4 binding and inhibiting B7-H6 expression.\",\n      \"method\": \"BRD4-specific inhibition, ChIP for BRD4/histone acetylation/P-TEFb at B7-H6 promoter/enhancer, flow cytometry, RT-PCR, metformin treatment, B7-H6 knockdown apoptosis assays\",\n      \"journal\": \"Oncoimmunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ChIP with multiple marks plus functional inhibition experiments in a single rigorous study\",\n      \"pmids\": [\"33796404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"B7-H6 contains six functional N-linked glycosylation sites; N43 and N208 glycosylation are conserved in jawed vertebrates and are essential for NK cell activation. Mechanistically, N43 glycosylation contributes to B7-H6/NKp30 binding affinity, while N208 glycosylation is required for membrane stability (lack of N208 leads to B7-H6 shedding from the membrane).\",\n      \"method\": \"PNGase F digestion, tunicamycin blockage, single-site mutagenesis, SDS-PAGE/Western blot, flow cytometry, NK cell activation assays, phylogenetic and structural analysis\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic site mutagenesis with in vitro functional validation and mechanistic dissection of individual glycan roles\",\n      \"pmids\": [\"38035117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"B7-H6 is strongly expressed on the surface of CD4+ T cells infected with HIV-2, and chronic NKp30 engagement by this surface B7-H6 leads to downregulation of NKp30 on NK cells, impairing their cytolytic function while increasing IFN-γ overproduction.\",\n      \"method\": \"Flow cytometry phenotyping of NK cells and target cells from HIV-2 patients, functional killing assays against B7-H6-expressing targets\",\n      \"journal\": \"AIDS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — patient cohort with functional assays; correlative but mechanistically linked to B7-H6 surface expression on infected cells\",\n      \"pmids\": [\"30325780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"B7-H6 promotes tumor cell proliferation, migration, and invasion in non-Hodgkin lymphoma via the Ras/MEK/ERK signaling pathway; B7-H6 knockdown inhibited Ras/MEK/ERK phosphorylation, and MEK inhibitor AZD8330 reversed migration/invasion induced by B7-H6.\",\n      \"method\": \"B7-H6 knockdown and overexpression in NHL cell lines, quantitative phosphoproteomics, Western blot for Ras/MEK/ERK pathway, MEK inhibitor rescue, in vivo xenograft model, CCK-8 and Transwell assays\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphoproteomics plus pathway inhibitor rescue plus in vivo validation\",\n      \"pmids\": [\"32606790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"B7-H6 preferentially expressed in glioma stem-like cells (GSLCs) promotes proliferation via the c-Myc/RNMT axis; B7-H6 knockdown decreased c-Myc expression, inhibited PI3K/Akt and ERK/MAPK signaling, and specifically reduced RNMT (RNA guanine-7 methyltransferase) expression among co-expressed genes. CRISPR-Cas9 knockout of B7-H6 in 293T cells also suppressed proliferation.\",\n      \"method\": \"siRNA knockdown, CRISPR-Cas9 knockout, Western blot for c-Myc/PI3K/Akt/ERK, TCGA co-expression analysis, flow cytometry, CCK-8 proliferation assays\",\n      \"journal\": \"Journal of immunology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO plus siRNA plus signaling pathway analysis; single lab\",\n      \"pmids\": [\"32322592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"B7-H6 undergoes nuclear translocation in T-lymphoblastic lymphoma (T-LBL) cells, with a specific nuclear localization sequence identified in the C-terminus; B7-H6 depletion in Jurkat cells impaired proliferation, migration, and invasion, and RNA-seq revealed differential expression of RAG-1 as a potential downstream mediator.\",\n      \"method\": \"Immunohistochemistry showing nuclear localization, nuclear localization sequence analysis, B7-H6 depletion, RNA sequencing, functional cell assays\",\n      \"journal\": \"Annals of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — localization finding with functional KD and RNAseq; single lab\",\n      \"pmids\": [\"33708955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SNRPA promotes B7-H6 expression in hepatocellular carcinoma by directly binding B7-H6 pre-mRNA and facilitating its maturation through intron 2 splicing; SNRPA-driven B7-H6 upregulation activates the B7-H6–STAT3/AKT signaling axis, promoting G1-S transition and inhibiting apoptosis, and confers resistance to lenvatinib.\",\n      \"method\": \"Nanopore full-length cDNA sequencing, RNA-binding protein immunoprecipitation sequencing, RIP-PCR with exon-exon and exon-intron junction primers, gain/loss-of-function experiments, Western blot for STAT3/AKT, flow cytometry, drug sensitivity assays\",\n      \"journal\": \"Bioscience trends\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA-binding/splicing mechanism with multiple orthogonal methods; single lab\",\n      \"pmids\": [\"40240166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"B7-H6 knockdown in AML (HEL-R) cells induces apoptosis, and high B7-H6 expression in AML activates anti-apoptotic signaling; the BRD4–B7-H6 regulatory axis is linked to the STAT3 pathway activity in these cells.\",\n      \"method\": \"B7-H6 knockdown (siRNA), flow cytometry for apoptosis, Western blot, correlation with BRD4 inhibition\",\n      \"journal\": \"Oncoimmunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional KD with apoptosis readout in same study as ChIP mechanistic work\",\n      \"pmids\": [\"33796404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NKp30-B7-H6 interaction in HBV-related acute-on-chronic liver failure leads to upregulation of IL-32 in NK cells; IL-32 in turn induces apoptosis of hepatocytes in a dose-dependent manner, establishing a mechanistic link between B7-H6 engagement and hepatocyte damage via IL-32.\",\n      \"method\": \"Co-culture of NK-92 cells with B7-H6-expressing hepatocytes, anti-NKp30 antibody stimulation, flow cytometry for IL-32 in NK cells, Annexin V/PI apoptosis assay of hepatocytes treated with IL-32, immunohistochemistry in patient liver samples\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro functional assays with receptor-blocking antibody and recombinant IL-32 dose-response; single lab\",\n      \"pmids\": [\"26241657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"atRA-induced downregulation of c-Myc reduces B7-H6 transcription in leukemia cells, rendering them resistant to NKp30/B7-H6-dependent NK cell killing; epigenetic modifications (DNA methylation, HDAC) were not responsible, as their inhibitors could not restore B7-H6 expression.\",\n      \"method\": \"CRISPR/Cas9 B7-H6 knockdown, siRNA c-Myc knockdown, atRA treatment, real-time PCR, flow cytometry, Western blot, NK cell cytotoxicity assays, HDAC/DNA methylation inhibitor controls\",\n      \"journal\": \"Cancer communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO plus siRNA plus pharmacological controls, functional NK readout; single lab\",\n      \"pmids\": [\"34236140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CRISPR-mediated knockout of B7-H6 in CD4+ T cells reduced NK cell responses to HIV-1-infected cells, confirming the NKp30:B7-H6 receptor–ligand pair as functionally required for NK recognition of HIV-infected cells.\",\n      \"method\": \"CRISPR editing of CD4+ T cells to knockout B7-H6, NK cell functional assays, NKp30 overexpression in NK cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with functional NK readout; preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2024.06.24.600449\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"B7-H6 promotes medulloblastoma cell migration and invasion by activating c-MYC expression, which in turn drives F-actin cytoskeletal rearrangement and upregulates MMP-2 and MMP-9.\",\n      \"method\": \"siRNA knockdown and plasmid overexpression of B7-H6, Transwell migration/invasion assays, Western blot for c-MYC/MMP-2/MMP-9, immunofluorescence staining for F-actin\",\n      \"journal\": \"Medical oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, KD/OE with downstream pathway markers; no reconstitution or ChIP\",\n      \"pmids\": [\"36692844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"G-CSF derived from gastric cancer tumors induces B7-H6 expression on neutrophils via NF-κB signaling pathway activation; B7-H6 on tumor-infiltrating neutrophils promotes their survival by blocking apoptosis, as B7-H6 blockade accelerated neutrophil apoptosis and shortened their lifespan.\",\n      \"method\": \"Flow cytometry phenotyping, in vitro neutrophil stimulation with G-CSF and tumor-conditioned supernatants, NF-κB pathway analysis, B7-H6 blockade with antibodies, apoptosis assays\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (stimulation + pathway inhibition + receptor blockade) with functional apoptosis readout\",\n      \"pmids\": [\"40054066\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NCR3LG1/B7-H6 is a tumor- and stress-induced cell-surface ligand belonging to the B7 family that binds directly to the activating NK cell receptor NKp30 (as defined by crystal structure of the complex), triggering NK cell cytotoxicity and cytokine secretion; its surface expression is transcriptionally driven by c-Myc and epigenetically regulated via BRD4/histone acetylation and HDAC activity at its promoter, post-translationally controlled by N-glycosylation at N43 (NKp30 binding) and N208 (membrane stability), and proteolytically shed from the cell surface by ADAM10/ADAM17 metalloproteases to generate immunosuppressive soluble B7-H6 that downregulates NKp30 on NK cells; intracellularly, B7-H6 promotes tumor cell-autonomous oncogenic signaling through c-Myc, Ras/MEK/ERK, PI3K/Akt, and STAT3 pathways to support proliferation, migration, invasion, and survival.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NCR3LG1 (B7-H6) is a B7-family cell-surface ligand that bridges innate immune surveillance and tumor-intrinsic oncogenic signaling. B7-H6 binds directly to the activating NK cell receptor NKp30 through CDR-like loops of its V-like domain, triggering NK cell cytotoxicity and cytokine secretion; this interaction is structurally distinct from canonical B7/CTLA-4 or PD-1/PD-L engagement [PMID:19528259, PMID:21422170]. Surface expression is transcriptionally driven by c-Myc binding at the B7-H6 promoter and epigenetically regulated via BRD4-mediated histone acetylation, while post-translational N-glycosylation at N43 tunes NKp30 binding affinity and at N208 controls membrane stability; ADAM10/ADAM17-mediated ectodomain shedding generates immunosuppressive soluble B7-H6 that downregulates NKp30 on NK cells [PMID:27622013, PMID:33796404, PMID:38035117, PMID:24780758]. Beyond its immune-ligand role, B7-H6 promotes tumor cell proliferation, migration, and survival through cell-autonomous activation of Ras/MEK/ERK, PI3K/Akt, and STAT3 pathways, and its expression on inflammatory monocytes, neutrophils, and virally infected cells extends its function to non-tumor immune contexts [PMID:32606790, PMID:32322592, PMID:23687088, PMID:40054066].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of B7-H6 as the long-sought cellular ligand for the orphan activating NK receptor NKp30 established the molecular basis for NKp30-dependent tumor immunosurveillance.\",\n      \"evidence\": \"Protein binding assays, functional NK cytotoxicity/cytokine assays, flow cytometry, and expression profiling across tumor vs. normal tissues\",\n      \"pmids\": [\"19528259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of interaction unknown\", \"Regulation of B7-H6 expression not addressed\", \"Whether soluble forms exist and their function unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Crystal structure of the NKp30–B7-H6 complex revealed an unconventional binding mode distinct from other B7-family immune checkpoint interactions, explaining NKp30's unique recognition strategy.\",\n      \"evidence\": \"X-ray crystallography of the NKp30–B7-H6 complex\",\n      \"pmids\": [\"21422170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of glycosylation in binding not resolved\", \"Whether B7-H6 dimerization is functionally relevant unclear\", \"Mechanism of signal transduction downstream of NKp30 engagement not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"B7-H6 expression was shown to extend beyond tumors to activated inflammatory myeloid cells, and its transcription was found to be epigenetically controlled through histone acetylation at its promoter, broadening understanding of B7-H6 regulation and its role in non-tumor immunity.\",\n      \"evidence\": \"In vitro stimulation of monocytes/neutrophils with TLR ligands and cytokines; HDAC inhibitor treatment, siRNA knockdown of HDAC2/3, ChIP for histone acetylation at B7-H6 promoter, luciferase reporters, functional NK assays\",\n      \"pmids\": [\"23687088\", \"23801635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific transcription factors driving B7-H6 promoter activity not identified\", \"In vivo relevance of myeloid B7-H6 in infection/inflammation not established\", \"Whether exosomal B7-H6 is functionally active on NK cells unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that ADAM10/ADAM17 metalloproteases shed B7-H6 ectodomain from tumor cells established that soluble B7-H6 functions as an immunosuppressive decoy impairing NKp30-mediated NK activation.\",\n      \"evidence\": \"Pharmacological ADAM inhibitors, siRNA knockdown of ADAM10/17, flow cytometry, NK activation assays, ELISA for soluble B7-H6 in patient sera\",\n      \"pmids\": [\"24780758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage site on B7-H6 not mapped\", \"Relative contributions of ADAM10 vs. ADAM17 in different tumor types not resolved\", \"Whether shedding is regulated by upstream signals unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Multiple studies demonstrated that therapeutic stress upregulates surface B7-H6 to sensitize tumor cells to NK lysis, while chronic B7-H6 exposure (surface or soluble) in ovarian carcinoma and neuroblastoma downregulates NKp30 to exhaust NK cells, revealing B7-H6's dual role as both immune activator and suppressor depending on context.\",\n      \"evidence\": \"Drug/radiation/heat-shock treatment of tumor lines with shRNA rescue; patient tumor-associated NK cell phenotyping from peritoneal fluids; patient serum functional inhibition assays with NKp30-blocking antibodies\",\n      \"pmids\": [\"26472927\", \"26137398\", \"25877893\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signaling pathways governing stress-induced B7-H6 upregulation not identified\", \"Threshold of B7-H6 exposure triggering NKp30 downregulation not quantified\", \"Whether NKp30 downregulation is reversible in vivo not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of c-Myc/N-Myc as direct transcriptional activators of B7-H6 provided the first specific transcription factor–promoter mechanism and linked oncogene-driven expression to NK immune recognition.\",\n      \"evidence\": \"ChIP showing c-Myc binding at B7-H6 promoter, luciferase reporter assays, siRNA/shRNA knockdown of c-Myc/N-Myc, NK degranulation assays across multiple tumor types\",\n      \"pmids\": [\"27622013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other oncogenic transcription factors contribute to B7-H6 expression not addressed\", \"Post-transcriptional regulation not explored\", \"Whether c-Myc also drives B7-H6 in non-tumor cells unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"HCMV proteins US18 and US20 were shown to downregulate B7-H6 via endosomal degradation, establishing a viral immune evasion mechanism targeting the NKp30–B7-H6 axis.\",\n      \"evidence\": \"Expression of individual HCMV genes in cells, flow cytometry, NK killing assays, endosomal/lysosomal degradation inhibitors\",\n      \"pmids\": [\"28819195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other herpesviruses target B7-H6 not tested\", \"Specific endosomal trafficking machinery involved not identified\", \"Whether US18/US20 directly bind B7-H6 not shown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"B7-H6 was found to have tumor cell-autonomous signaling functions, promoting proliferation, migration, and invasion through Ras/MEK/ERK and PI3K/Akt/c-Myc pathways independent of its NK ligand role.\",\n      \"evidence\": \"B7-H6 knockdown/overexpression in NHL and glioma cell lines, phosphoproteomics, MEK inhibitor rescue, Western blot for pathway components, xenograft models, CRISPR-Cas9 KO in 293T cells\",\n      \"pmids\": [\"32606790\", \"32322592\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether B7-H6 signals through a cis-receptor or acts receptor-independently is unknown\", \"No binding partner on the tumor cell surface identified for intrinsic signaling\", \"Single-lab findings for each tumor type\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"BRD4 was identified as an epigenetic reader that activates B7-H6 transcription by recognizing acetylated histones at the promoter and recruiting P-TEFb; additionally, atRA-mediated c-Myc downregulation was shown to reduce B7-H6 and confer NK resistance, unifying transcriptional and epigenetic control.\",\n      \"evidence\": \"ChIP for BRD4, histone acetylation, and P-TEFb at B7-H6 promoter/enhancer; BRD4 inhibition; metformin treatment; atRA treatment with CRISPR/siRNA controls and NK cytotoxicity assays\",\n      \"pmids\": [\"33796404\", \"34236140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether BRD4 and c-Myc cooperate at the B7-H6 promoter or act independently not resolved\", \"Enhancer–promoter looping mechanism not demonstrated\", \"In vivo therapeutic validation of BRD4/c-Myc targeting to modulate B7-H6 lacking\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Systematic glycosylation mapping revealed that conserved N-linked glycans at N43 and N208 serve distinct mechanistic roles—N43 for NKp30 binding affinity and N208 for membrane retention—explaining how post-translational modification fine-tunes both immune recognition and shedding.\",\n      \"evidence\": \"PNGase F digestion, tunicamycin treatment, single-site mutagenesis of six N-glycosylation sites, flow cytometry, NK activation assays, phylogenetic analysis\",\n      \"pmids\": [\"38035117\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Glycan composition at each site not determined\", \"Whether glycosylation status changes under stress or in different tumor types unknown\", \"How N208 glycosylation mechanistically prevents shedding not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"SNRPA was identified as an RNA-binding protein that promotes B7-H6 pre-mRNA maturation via intron 2 splicing, connecting RNA processing to B7-H6-driven STAT3/AKT signaling and lenvatinib resistance in hepatocellular carcinoma; separately, G-CSF-induced NF-κB signaling was shown to drive B7-H6 on tumor-infiltrating neutrophils to promote their survival.\",\n      \"evidence\": \"Nanopore sequencing, RIP-seq/RIP-PCR with exon-junction primers, gain/loss-of-function for SNRPA, Western blot; G-CSF stimulation, NF-κB pathway analysis, B7-H6 antibody blockade, apoptosis assays\",\n      \"pmids\": [\"40240166\", \"40054066\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SNRPA–B7-H6 splicing mechanism needs independent validation\", \"Whether SNRPA regulation of B7-H6 extends beyond HCC not tested\", \"How B7-H6 blocks neutrophil apoptosis mechanistically (cis-signaling vs. receptor engagement) remains undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The mechanism by which B7-H6 transduces cell-autonomous oncogenic signals (Ras/MEK/ERK, PI3K/Akt, STAT3) remains unknown—no cis-receptor, intracellular binding partner, or signaling domain has been identified to explain tumor-intrinsic B7-H6 function.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No intracellular binding partners identified\", \"No signaling motif in B7-H6 cytoplasmic tail characterized\", \"Relationship between nuclear-localized B7-H6 and transcriptional effects unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [15, 16, 18, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 3, 7, 13]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [2, 8, 9, 11]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2, 8, 9, 10, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [15, 16, 18, 24]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15, 16, 18, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NCR3\",\n      \"ADAM10\",\n      \"ADAM17\",\n      \"MYC\",\n      \"BRD4\",\n      \"SNRPA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}