{"gene":"WDFY4","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2018,"finding":"WDFY4 is essential for cross-presentation of cell-associated antigens by Batf3-dependent cDC1s (CD8α+/XCR1+ classical dendritic cells) to prime CD8+ T cells in vivo; Wdfy4-/- mice have morphologically and functionally normal cDC1 populations capable of IL-12 production and Toxoplasma gondii protection, but fail to cross-present cell-associated antigens, prime virus-specific CD8+ T cells, or induce tumor rejection. WDFY4 is not required for MHC class II presentation or cross-presentation by monocyte-derived DCs.","method":"CRISPR functional screen; Wdfy4-/- mouse knockout with in vivo antigen presentation assays, viral CD8+ T cell priming, tumor rejection assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple orthogonal functional readouts (cross-presentation, CD8+ T cell priming, tumor rejection, infection resistance), replicated across multiple antigen systems","pmids":["30409884"],"is_preprint":false},{"year":2025,"finding":"WDFY4-dependent cross-presentation is not restricted to cDC1s: for immune complex antigens, either cDC1 or cDC2 can perform cross-presentation to CD8+ T cells, and this cDC2-mediated cross-presentation is also WDFY4 dependent. Monocyte-derived DCs cannot substitute. Mice lacking cDC1 but vaccinated with immune complexes can cross-prime CD8+ T cells sufficient for tumor rejection via cDC2, in a WDFY4-dependent manner.","method":"Genetic models (cDC1-deficient and Wdfy4-/- mice); in vivo antigen presentation assays with immune complex and cell-associated antigens; tumor rejection assays","journal":"The Journal of Experimental Medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models with orthogonal functional readouts (cross-presentation, tumor rejection) across distinct antigen types in vivo","pmids":["39918736"],"is_preprint":false},{"year":2023,"finding":"In NOD mice, WDFY4 deficiency (via CRISPR/Cas9) abolishes cDC1 cross-presentation of cell-associated antigens to prime autoreactive CD8+ T cells, prevents progression of autoimmune diabetes beyond peri-islet inflammation, and blocks recruitment of autoreactive CD4+ T cells into islets, while MHC-II antigen presentation and β cell-specific CD4+ T cell activation in lymph nodes remain intact.","method":"CRISPR/Cas9 knockout in NOD mice; diabetes incidence monitoring; CD4+ and CD8+ T cell priming assays; histology of islet inflammation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic model with multiple orthogonal disease and cellular readouts, internally controlled with heterozygous mice","pmids":["36940342"],"is_preprint":false},{"year":2018,"finding":"WDFY4 and its truncated isoform (tr-WDFY4, generated by a splicing variant associated with CADM risk allele) interact with pattern recognition receptors TLR3, TLR4, TLR9, and MDA5, and augment NF-κB activation downstream of these receptors. Both isoforms also enhance MDA5-induced apoptosis, with tr-WDFY4 showing greater enhancement of apoptosis.","method":"In vitro co-immunoprecipitation/interaction assays; reporter assays for NF-κB activation; apoptosis assays in transfected cells; trans-eQTL analysis","journal":"Annals of the Rheumatic Diseases","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP/interaction and reporter assays in a single lab, two orthogonal methods (binding + functional NF-κB/apoptosis readouts)","pmids":["29331962"],"is_preprint":false},{"year":2018,"finding":"B cell-conditional knockout of Wdfy4 in mice reduces total B cell numbers and multiple B cell subpopulations in the periphery, causes a defect in the pro- to pre-B cell transition in bone marrow, impairs antibody responses to antigen challenge, and alleviates SLE phenotypes (reduced autoantibody production and glomerulonephritis). WDFY4 loss in B cells increases LC3 lipidation independently of p62 and Beclin1, indicating a role in facilitating noncanonical autophagy.","method":"B cell-conditional Wdfy4 knockout mice; flow cytometry of B cell subsets; antibody response assays; pristane-induced SLE model; LC3 lipidation assay; p62/Beclin1 independence tested","journal":"Journal of Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout with multiple cellular and biochemical readouts in a single lab","pmids":["30257884"],"is_preprint":false},{"year":2021,"finding":"T cell-specific deficiency of Wdfy4 in mice reduces peripheral CD8+ T cell numbers, promotes tumor growth when challenged with transplantable tumors, enhances apoptosis of CD8+ T cells, increases intracellular reactive oxygen species with upregulation of Nox2, and is mechanistically associated with activation of the p53 pathway and inhibition of the ERK pathway. WDFY4 also participates in T cell proliferation.","method":"T cell-conditional Wdfy4 knockout mice; tumor challenge experiments; flow cytometry; ROS measurement; p53 and ERK pathway analysis","journal":"Molecular Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout with multiple orthogonal cellular and biochemical readouts in a single lab","pmids":["34482201"],"is_preprint":false},{"year":2021,"finding":"WDFY4 deficiency in mice promotes Th2 cell differentiation and Th2 cytokine production from naïve CD4+ T cells differentiated in vitro, and exacerbates ovalbumin-induced asthma in vivo with higher Th2 cytokines, increased inflammatory cell infiltration, goblet cell hyperplasia, mucus production, and collagen deposition.","method":"Wdfy4-knockout mice; in vitro Th2 differentiation assay from naïve CD4+ T cells; OVA-induced asthma model; cytokine measurement; histology","journal":"International Archives of Allergy and Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout model with in vitro and in vivo orthogonal readouts in a single lab","pmids":["34425575"],"is_preprint":false},{"year":2012,"finding":"The transcription factor YY1 (Yin Yang 1) binds to intronic WDFY4 variant rs877819; the risk allele A has lower YY1 binding affinity compared to the G allele, resulting in reduced WDFY4 transcriptional activity. YY1 knockdown reduces WDFY4 expression, while YY1 overexpression increases it, and ChIP confirms YY1 occupancy at this site.","method":"Electrophoretic mobility shift assay (EMSA); supershift assay; dual-luciferase reporter assay; YY1 siRNA knockdown; YY1 overexpression; chromatin immunoprecipitation (ChIP)","journal":"Genes and Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (EMSA, supershift, luciferase, ChIP, KD/OE) in a single lab","pmids":["22972472"],"is_preprint":false},{"year":2025,"finding":"WDFY4 interacts with LAPTM5 (lysosomal transmembrane protein 5), validated by co-immunoprecipitation and immunofluorescence co-localization. WDFY4 knockdown inhibits LAPTM5 expression and activates the downstream CDC42/mTOR/4EBP1/SLC7A11 pathway. LAPTM5 overexpression or CDC42 inhibition rescues WDFY4 knockdown-mediated suppression of ferroptosis in endothelial cells, placing WDFY4 upstream of LAPTM5 in a ferroptosis-promoting pathway in atherosclerosis.","method":"Co-immunoprecipitation; immunofluorescence co-localization; endothelium-specific transgenic/knockout mice; pathway analysis (CDC42/mTOR/4EBP1/SLC7A11); rescue experiments with LAPTM5 overexpression and CDC42 inhibitor ML141","journal":"Journal of Cellular and Molecular Medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and co-localization with functional rescue experiments in a single lab; mechanistic pathway partially inferred","pmids":["40755163"],"is_preprint":false}],"current_model":"WDFY4, a BEACH domain-containing protein, is essential for cross-presentation of cell-associated and immune complex antigens by both cDC1 and cDC2 classical dendritic cells to prime CD8+ T cells (without affecting MHC-II presentation or cDC1 development), and also regulates B cell development and noncanonical autophagy, CD8+ T cell survival via ROS/p53/ERK pathways, and Th2 cell differentiation; in innate signaling, WDFY4 physically interacts with TLR3/4/9 and MDA5 to augment NF-κB activation, and in endothelial cells promotes ferroptosis through a LAPTM5/CDC42/mTOR/4EBP1/SLC7A11 pathway, with its transcription regulated by YY1 binding to a disease-associated intronic variant."},"narrative":{"mechanistic_narrative":"WDFY4 is a BEACH domain-containing protein that serves as an essential, non-redundant intracellular factor for the cross-presentation of exogenous antigens by classical dendritic cells, enabling priming of CD8+ T cells [PMID:30409884]. In Batf3-dependent cDC1s, WDFY4 is dispensable for cell development, IL-12 production, and MHC class II presentation, but is specifically required to cross-present cell-associated antigens, prime virus-specific CD8+ T cells, and drive tumor rejection [PMID:30409884]. This requirement extends beyond cDC1s: cross-presentation of immune complex antigens by cDC2s is likewise WDFY4-dependent, whereas monocyte-derived DCs cannot substitute in either case [PMID:39918736]. The same pathway operates in autoimmunity, where WDFY4-dependent cDC1 cross-presentation is needed to prime autoreactive CD8+ T cells and drive progression of autoimmune diabetes [PMID:36940342]. Beyond dendritic cells, WDFY4 acts intrinsically in other immune compartments: it promotes the pro- to pre-B cell transition and antibody responses while facilitating a p62/Beclin1-independent noncanonical autophagy marked by LC3 lipidation [PMID:30257884], supports CD8+ T cell survival and proliferation by restraining ROS/Nox2, p53, and ERK signaling [PMID:34482201], and limits Th2 cell differentiation [PMID:34425575]. In innate signaling, WDFY4 and a truncated isoform physically interact with the pattern recognition receptors TLR3, TLR4, TLR9, and MDA5 to augment NF-κB activation and MDA5-induced apoptosis [PMID:29331962]. WDFY4 also interacts with LAPTM5 to promote endothelial ferroptosis via a downstream CDC42/mTOR/4EBP1/SLC7A11 axis [PMID:40755163]. WDFY4 transcription is controlled by YY1 binding at an intronic disease-associated variant, where the risk allele lowers YY1 affinity and reduces expression [PMID:22972472].","teleology":[{"year":2012,"claim":"Established how a disease-associated noncoding WDFY4 variant alters gene function, showing it is a transcriptional regulatory variant rather than a coding change.","evidence":"EMSA, supershift, dual-luciferase, YY1 knockdown/overexpression, and ChIP at intronic variant rs877819","pmids":["22972472"],"confidence":"Medium","gaps":["Does not connect altered WDFY4 expression level to a specific cellular phenotype","Single-lab characterization of one variant","No demonstration that YY1 regulation operates in the relevant immune cell types"]},{"year":2018,"claim":"Defined WDFY4 as the non-redundant intracellular machinery required specifically for cDC1 cross-presentation, separating this function from cDC1 development and MHC-II presentation.","evidence":"CRISPR functional screen and Wdfy4-/- mice with in vivo cross-presentation, viral CD8+ priming, and tumor rejection assays","pmids":["30409884"],"confidence":"High","gaps":["Molecular mechanism by which WDFY4 enables antigen cross-presentation not resolved","No biochemical partners or subcellular trafficking step identified","Role of the BEACH domain in this function untested"]},{"year":2018,"claim":"Linked WDFY4 to innate signaling and connected it to autoimmune disease risk by showing direct association with pattern recognition receptors and modulation of downstream responses.","evidence":"Co-IP/interaction assays, NF-κB reporter and apoptosis assays in transfected cells, and trans-eQTL analysis for full-length and truncated isoforms","pmids":["29331962"],"confidence":"Medium","gaps":["Co-IP/reporter assays in a single lab without reciprocal in vivo validation","Interaction domains and mechanism of NF-κB augmentation not mapped","Relationship between this innate signaling role and the cross-presentation role unclear"]},{"year":2018,"claim":"Extended WDFY4 function to B cell development and antibody responses and tied it mechanistically to a noncanonical autophagy process.","evidence":"B cell-conditional Wdfy4 knockout mice, flow cytometry, antibody and SLE-model assays, and LC3 lipidation with p62/Beclin1 independence testing","pmids":["30257884"],"confidence":"Medium","gaps":["Molecular basis of the noncanonical autophagy contribution not defined","Single-lab conditional knockout","Connection to the cross-presentation pathway not established"]},{"year":2021,"claim":"Identified a T cell-intrinsic role for WDFY4 in CD8+ T cell survival and proliferation, implicating ROS, p53, and ERK signaling.","evidence":"T cell-conditional Wdfy4 knockout mice with tumor challenge, ROS/Nox2 measurement, and p53/ERK pathway analysis","pmids":["34482201"],"confidence":"Medium","gaps":["Causal ordering between WDFY4, ROS, p53, and ERK not dissected","No direct molecular target of WDFY4 identified","Single-lab study"]},{"year":2021,"claim":"Showed WDFY4 restrains Th2 differentiation, broadening its immune role to allergic inflammation.","evidence":"Wdfy4-knockout mice with in vitro Th2 differentiation and OVA-induced asthma model with cytokine and histological readouts","pmids":["34425575"],"confidence":"Medium","gaps":["Mechanism by which WDFY4 limits Th2 polarization not defined","Cell-intrinsic versus extrinsic contribution not fully resolved","Single-lab study"]},{"year":2025,"claim":"Demonstrated that WDFY4-dependent cross-presentation is not cDC1-restricted, establishing cDC2 as a sufficient cross-presenting cell for immune complex antigens.","evidence":"cDC1-deficient and Wdfy4-/- mice with in vivo immune complex/cell-associated antigen presentation and tumor rejection assays","pmids":["39918736"],"confidence":"High","gaps":["Molecular determinant of which antigens route through cDC2 versus cDC1 unknown","Whether WDFY4 acts identically in cDC1 and cDC2 not established at the mechanistic level"]},{"year":2025,"claim":"Placed WDFY4 in an endothelial ferroptosis pathway through a direct interaction with LAPTM5 upstream of a CDC42/mTOR/4EBP1/SLC7A11 axis.","evidence":"Co-IP, immunofluorescence co-localization, endothelium-specific mouse models, and rescue experiments with LAPTM5 overexpression and CDC42 inhibition","pmids":["40755163"],"confidence":"Medium","gaps":["Downstream pathway partially inferred rather than fully demonstrated","Single-lab Co-IP without reciprocal validation","Relationship of this endothelial role to immune cross-presentation function unknown"]},{"year":null,"claim":"The core biochemical mechanism by which WDFY4 enables antigen cross-presentation, and how its BEACH domain and diverse interaction partners coordinate this with its other immune and ferroptotic roles, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model or defined enzymatic/scaffolding activity for WDFY4","No identified molecular step in the cross-presentation pathway that WDFY4 executes","Unclear whether the distinct reported functions reflect one unifying mechanism or context-specific roles"]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,2,3,4,5,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,8]}],"complexes":[],"partners":["TLR3","TLR4","TLR9","MDA5","LAPTM5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6ZS81","full_name":"WD repeat- and FYVE domain-containing protein 4","aliases":[],"length_aa":3184,"mass_kda":353.6,"function":"Plays a critical role in the regulation of cDC1-mediated cross-presentation of viral and tumor antigens in dendritic cells. Mechanistically, acts near the plasma membrane and interacts with endosomal membranes to promote endosomal-to-cytosol antigen trafficking. Also plays a role in B-cell survival through regulation of autophagy","subcellular_location":"Early endosome; Endoplasmic reticulum","url":"https://www.uniprot.org/uniprotkb/Q6ZS81/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/WDFY4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/WDFY4","total_profiled":1310},"omim":[{"mim_id":"613316","title":"WD REPEAT- AND FYVE DOMAIN-CONTAINING PROTEIN 4; WDFY4","url":"https://www.omim.org/entry/613316"},{"mim_id":"606493","title":"EXOSOME COMPONENT 1; EXOSC1","url":"https://www.omim.org/entry/606493"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":11.0},{"tissue":"lymphoid tissue","ntpm":26.6}],"url":"https://www.proteinatlas.org/search/WDFY4"},"hgnc":{"alias_symbol":["KIAA1607","Em:AC060234.3","FLJ45748"],"prev_symbol":["C10orf64"]},"alphafold":{"accession":"Q6ZS81","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZS81","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZS81-3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZS81-3-F1-predicted_aligned_error_v6.png","plddt_mean":78.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=WDFY4","jax_strain_url":"https://www.jax.org/strain/search?query=WDFY4"},"sequence":{"accession":"Q6ZS81","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6ZS81.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6ZS81/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZS81"}},"corpus_meta":[{"pmid":"20169177","id":"PMC_20169177","title":"Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus.","date":"2010","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20169177","citation_count":345,"is_preprint":false},{"pmid":"30409884","id":"PMC_30409884","title":"WDFY4 is required for cross-presentation in response to viral and tumor antigens.","date":"2018","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/30409884","citation_count":270,"is_preprint":false},{"pmid":"29331962","id":"PMC_29331962","title":"Splicing variant of WDFY4 augments MDA5 signalling and the risk of clinically amyopathic dermatomyositis.","date":"2018","source":"Annals of the rheumatic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/29331962","citation_count":64,"is_preprint":false},{"pmid":"22972472","id":"PMC_22972472","title":"An intronic variant associated with systemic lupus erythematosus changes the binding affinity of Yinyang1 to downregulate WDFY4.","date":"2012","source":"Genes and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/22972472","citation_count":45,"is_preprint":false},{"pmid":"30257884","id":"PMC_30257884","title":"WDFY4 Is Involved in Symptoms of Systemic Lupus Erythematosus by Modulating B Cell Fate via Noncanonical Autophagy.","date":"2018","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/30257884","citation_count":28,"is_preprint":false},{"pmid":"36637178","id":"PMC_36637178","title":"WDFY4 polymorphisms in Chinese patients with anti-MDA5 dermatomyositis is associated with rapid progressive interstitial lung disease.","date":"2023","source":"Rheumatology (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/36637178","citation_count":21,"is_preprint":false},{"pmid":"24549174","id":"PMC_24549174","title":"E26 transformation-specific-1 (ETS1) and WDFY family member 4 (WDFY4) polymorphisms in Chinese patients with rheumatoid arthritis.","date":"2014","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/24549174","citation_count":15,"is_preprint":false},{"pmid":"34482201","id":"PMC_34482201","title":"Deficiency in WDFY4 reduces the number of CD8+ T cells via reactive oxygen species-induced apoptosis.","date":"2021","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34482201","citation_count":14,"is_preprint":false},{"pmid":"39918736","id":"PMC_39918736","title":"Shared pathway of WDFY4-dependent cross-presentation of immune complexes by cDC1 and cDC2.","date":"2025","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39918736","citation_count":13,"is_preprint":false},{"pmid":"36940342","id":"PMC_36940342","title":"WDFY4 deficiency in NOD mice ameliorates autoimmune diabetes and insulitis.","date":"2023","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/36940342","citation_count":9,"is_preprint":false},{"pmid":"38507703","id":"PMC_38507703","title":"The clinical relevance of WDFY4 in autoimmune diseases in diverse ancestral populations.","date":"2024","source":"Rheumatology (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/38507703","citation_count":9,"is_preprint":false},{"pmid":"37728413","id":"PMC_37728413","title":"Ferroptosis and WDFY4 as novel targets for immunotherapy of lung adenocarcinoma.","date":"2023","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/37728413","citation_count":8,"is_preprint":false},{"pmid":"34425575","id":"PMC_34425575","title":"Lack of WDFY4 Aggravates Ovalbumin-Induced Asthma via Enhanced Th2 Cell Differentiation.","date":"2021","source":"International archives of allergy and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34425575","citation_count":7,"is_preprint":false},{"pmid":"33189404","id":"PMC_33189404","title":"BVVL/ FL: features caused by SLC52A3 mutations; WDFY4 and TNFSF13B may be novel causative genes.","date":"2020","source":"Neurobiology of aging","url":"https://pubmed.ncbi.nlm.nih.gov/33189404","citation_count":7,"is_preprint":false},{"pmid":"40755163","id":"PMC_40755163","title":"WDFY4 Promotes the Progression of Atherosclerosis by Regulating Ferroptosis Mediated by the LAPTM5/CDC42/mTOR/4EBP1/SLC7A11 Pathway.","date":"2025","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40755163","citation_count":2,"is_preprint":false},{"pmid":"42169052","id":"PMC_42169052","title":"WDFY4 gene polymorphisms (rs11101565 and rs2671702) are not associated with breast cancer risk in Bangladeshi women: a case-control study.","date":"2026","source":"BMC research notes","url":"https://pubmed.ncbi.nlm.nih.gov/42169052","citation_count":0,"is_preprint":false},{"pmid":"41965494","id":"PMC_41965494","title":"Epigenetic alterations in WDFY4 DNA methylation are linked to immunoinflammatory processes in rheumatoid arthritis: insights from a case-control study.","date":"2026","source":"Clinical rheumatology","url":"https://pubmed.ncbi.nlm.nih.gov/41965494","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11069,"output_tokens":2777,"usd":0.037431,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10072,"output_tokens":3325,"usd":0.066742,"stage2_stop_reason":"end_turn"},"total_usd":0.104173,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"WDFY4 is essential for cross-presentation of cell-associated antigens by Batf3-dependent cDC1s (CD8α+/XCR1+ classical dendritic cells) to prime CD8+ T cells in vivo; Wdfy4-/- mice have morphologically and functionally normal cDC1 populations capable of IL-12 production and Toxoplasma gondii protection, but fail to cross-present cell-associated antigens, prime virus-specific CD8+ T cells, or induce tumor rejection. WDFY4 is not required for MHC class II presentation or cross-presentation by monocyte-derived DCs.\",\n      \"method\": \"CRISPR functional screen; Wdfy4-/- mouse knockout with in vivo antigen presentation assays, viral CD8+ T cell priming, tumor rejection assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple orthogonal functional readouts (cross-presentation, CD8+ T cell priming, tumor rejection, infection resistance), replicated across multiple antigen systems\",\n      \"pmids\": [\"30409884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"WDFY4-dependent cross-presentation is not restricted to cDC1s: for immune complex antigens, either cDC1 or cDC2 can perform cross-presentation to CD8+ T cells, and this cDC2-mediated cross-presentation is also WDFY4 dependent. Monocyte-derived DCs cannot substitute. Mice lacking cDC1 but vaccinated with immune complexes can cross-prime CD8+ T cells sufficient for tumor rejection via cDC2, in a WDFY4-dependent manner.\",\n      \"method\": \"Genetic models (cDC1-deficient and Wdfy4-/- mice); in vivo antigen presentation assays with immune complex and cell-associated antigens; tumor rejection assays\",\n      \"journal\": \"The Journal of Experimental Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models with orthogonal functional readouts (cross-presentation, tumor rejection) across distinct antigen types in vivo\",\n      \"pmids\": [\"39918736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In NOD mice, WDFY4 deficiency (via CRISPR/Cas9) abolishes cDC1 cross-presentation of cell-associated antigens to prime autoreactive CD8+ T cells, prevents progression of autoimmune diabetes beyond peri-islet inflammation, and blocks recruitment of autoreactive CD4+ T cells into islets, while MHC-II antigen presentation and β cell-specific CD4+ T cell activation in lymph nodes remain intact.\",\n      \"method\": \"CRISPR/Cas9 knockout in NOD mice; diabetes incidence monitoring; CD4+ and CD8+ T cell priming assays; histology of islet inflammation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic model with multiple orthogonal disease and cellular readouts, internally controlled with heterozygous mice\",\n      \"pmids\": [\"36940342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WDFY4 and its truncated isoform (tr-WDFY4, generated by a splicing variant associated with CADM risk allele) interact with pattern recognition receptors TLR3, TLR4, TLR9, and MDA5, and augment NF-κB activation downstream of these receptors. Both isoforms also enhance MDA5-induced apoptosis, with tr-WDFY4 showing greater enhancement of apoptosis.\",\n      \"method\": \"In vitro co-immunoprecipitation/interaction assays; reporter assays for NF-κB activation; apoptosis assays in transfected cells; trans-eQTL analysis\",\n      \"journal\": \"Annals of the Rheumatic Diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP/interaction and reporter assays in a single lab, two orthogonal methods (binding + functional NF-κB/apoptosis readouts)\",\n      \"pmids\": [\"29331962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"B cell-conditional knockout of Wdfy4 in mice reduces total B cell numbers and multiple B cell subpopulations in the periphery, causes a defect in the pro- to pre-B cell transition in bone marrow, impairs antibody responses to antigen challenge, and alleviates SLE phenotypes (reduced autoantibody production and glomerulonephritis). WDFY4 loss in B cells increases LC3 lipidation independently of p62 and Beclin1, indicating a role in facilitating noncanonical autophagy.\",\n      \"method\": \"B cell-conditional Wdfy4 knockout mice; flow cytometry of B cell subsets; antibody response assays; pristane-induced SLE model; LC3 lipidation assay; p62/Beclin1 independence tested\",\n      \"journal\": \"Journal of Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout with multiple cellular and biochemical readouts in a single lab\",\n      \"pmids\": [\"30257884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"T cell-specific deficiency of Wdfy4 in mice reduces peripheral CD8+ T cell numbers, promotes tumor growth when challenged with transplantable tumors, enhances apoptosis of CD8+ T cells, increases intracellular reactive oxygen species with upregulation of Nox2, and is mechanistically associated with activation of the p53 pathway and inhibition of the ERK pathway. WDFY4 also participates in T cell proliferation.\",\n      \"method\": \"T cell-conditional Wdfy4 knockout mice; tumor challenge experiments; flow cytometry; ROS measurement; p53 and ERK pathway analysis\",\n      \"journal\": \"Molecular Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout with multiple orthogonal cellular and biochemical readouts in a single lab\",\n      \"pmids\": [\"34482201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WDFY4 deficiency in mice promotes Th2 cell differentiation and Th2 cytokine production from naïve CD4+ T cells differentiated in vitro, and exacerbates ovalbumin-induced asthma in vivo with higher Th2 cytokines, increased inflammatory cell infiltration, goblet cell hyperplasia, mucus production, and collagen deposition.\",\n      \"method\": \"Wdfy4-knockout mice; in vitro Th2 differentiation assay from naïve CD4+ T cells; OVA-induced asthma model; cytokine measurement; histology\",\n      \"journal\": \"International Archives of Allergy and Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout model with in vitro and in vivo orthogonal readouts in a single lab\",\n      \"pmids\": [\"34425575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The transcription factor YY1 (Yin Yang 1) binds to intronic WDFY4 variant rs877819; the risk allele A has lower YY1 binding affinity compared to the G allele, resulting in reduced WDFY4 transcriptional activity. YY1 knockdown reduces WDFY4 expression, while YY1 overexpression increases it, and ChIP confirms YY1 occupancy at this site.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA); supershift assay; dual-luciferase reporter assay; YY1 siRNA knockdown; YY1 overexpression; chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"Genes and Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (EMSA, supershift, luciferase, ChIP, KD/OE) in a single lab\",\n      \"pmids\": [\"22972472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"WDFY4 interacts with LAPTM5 (lysosomal transmembrane protein 5), validated by co-immunoprecipitation and immunofluorescence co-localization. WDFY4 knockdown inhibits LAPTM5 expression and activates the downstream CDC42/mTOR/4EBP1/SLC7A11 pathway. LAPTM5 overexpression or CDC42 inhibition rescues WDFY4 knockdown-mediated suppression of ferroptosis in endothelial cells, placing WDFY4 upstream of LAPTM5 in a ferroptosis-promoting pathway in atherosclerosis.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence co-localization; endothelium-specific transgenic/knockout mice; pathway analysis (CDC42/mTOR/4EBP1/SLC7A11); rescue experiments with LAPTM5 overexpression and CDC42 inhibitor ML141\",\n      \"journal\": \"Journal of Cellular and Molecular Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and co-localization with functional rescue experiments in a single lab; mechanistic pathway partially inferred\",\n      \"pmids\": [\"40755163\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WDFY4, a BEACH domain-containing protein, is essential for cross-presentation of cell-associated and immune complex antigens by both cDC1 and cDC2 classical dendritic cells to prime CD8+ T cells (without affecting MHC-II presentation or cDC1 development), and also regulates B cell development and noncanonical autophagy, CD8+ T cell survival via ROS/p53/ERK pathways, and Th2 cell differentiation; in innate signaling, WDFY4 physically interacts with TLR3/4/9 and MDA5 to augment NF-κB activation, and in endothelial cells promotes ferroptosis through a LAPTM5/CDC42/mTOR/4EBP1/SLC7A11 pathway, with its transcription regulated by YY1 binding to a disease-associated intronic variant.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WDFY4 is a BEACH domain-containing protein that serves as an essential, non-redundant intracellular factor for the cross-presentation of exogenous antigens by classical dendritic cells, enabling priming of CD8+ T cells [#0]. In Batf3-dependent cDC1s, WDFY4 is dispensable for cell development, IL-12 production, and MHC class II presentation, but is specifically required to cross-present cell-associated antigens, prime virus-specific CD8+ T cells, and drive tumor rejection [#0]. This requirement extends beyond cDC1s: cross-presentation of immune complex antigens by cDC2s is likewise WDFY4-dependent, whereas monocyte-derived DCs cannot substitute in either case [#1]. The same pathway operates in autoimmunity, where WDFY4-dependent cDC1 cross-presentation is needed to prime autoreactive CD8+ T cells and drive progression of autoimmune diabetes [#2]. Beyond dendritic cells, WDFY4 acts intrinsically in other immune compartments: it promotes the pro- to pre-B cell transition and antibody responses while facilitating a p62/Beclin1-independent noncanonical autophagy marked by LC3 lipidation [#4], supports CD8+ T cell survival and proliferation by restraining ROS/Nox2, p53, and ERK signaling [#5], and limits Th2 cell differentiation [#6]. In innate signaling, WDFY4 and a truncated isoform physically interact with the pattern recognition receptors TLR3, TLR4, TLR9, and MDA5 to augment NF-\\u03baB activation and MDA5-induced apoptosis [#3]. WDFY4 also interacts with LAPTM5 to promote endothelial ferroptosis via a downstream CDC42/mTOR/4EBP1/SLC7A11 axis [#8]. WDFY4 transcription is controlled by YY1 binding at an intronic disease-associated variant, where the risk allele lowers YY1 affinity and reduces expression [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established how a disease-associated noncoding WDFY4 variant alters gene function, showing it is a transcriptional regulatory variant rather than a coding change.\",\n      \"evidence\": \"EMSA, supershift, dual-luciferase, YY1 knockdown/overexpression, and ChIP at intronic variant rs877819\",\n      \"pmids\": [\"22972472\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Does not connect altered WDFY4 expression level to a specific cellular phenotype\",\n        \"Single-lab characterization of one variant\",\n        \"No demonstration that YY1 regulation operates in the relevant immune cell types\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined WDFY4 as the non-redundant intracellular machinery required specifically for cDC1 cross-presentation, separating this function from cDC1 development and MHC-II presentation.\",\n      \"evidence\": \"CRISPR functional screen and Wdfy4-/- mice with in vivo cross-presentation, viral CD8+ priming, and tumor rejection assays\",\n      \"pmids\": [\"30409884\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Molecular mechanism by which WDFY4 enables antigen cross-presentation not resolved\",\n        \"No biochemical partners or subcellular trafficking step identified\",\n        \"Role of the BEACH domain in this function untested\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked WDFY4 to innate signaling and connected it to autoimmune disease risk by showing direct association with pattern recognition receptors and modulation of downstream responses.\",\n      \"evidence\": \"Co-IP/interaction assays, NF-\\u03baB reporter and apoptosis assays in transfected cells, and trans-eQTL analysis for full-length and truncated isoforms\",\n      \"pmids\": [\"29331962\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Co-IP/reporter assays in a single lab without reciprocal in vivo validation\",\n        \"Interaction domains and mechanism of NF-\\u03baB augmentation not mapped\",\n        \"Relationship between this innate signaling role and the cross-presentation role unclear\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended WDFY4 function to B cell development and antibody responses and tied it mechanistically to a noncanonical autophagy process.\",\n      \"evidence\": \"B cell-conditional Wdfy4 knockout mice, flow cytometry, antibody and SLE-model assays, and LC3 lipidation with p62/Beclin1 independence testing\",\n      \"pmids\": [\"30257884\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Molecular basis of the noncanonical autophagy contribution not defined\",\n        \"Single-lab conditional knockout\",\n        \"Connection to the cross-presentation pathway not established\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a T cell-intrinsic role for WDFY4 in CD8+ T cell survival and proliferation, implicating ROS, p53, and ERK signaling.\",\n      \"evidence\": \"T cell-conditional Wdfy4 knockout mice with tumor challenge, ROS/Nox2 measurement, and p53/ERK pathway analysis\",\n      \"pmids\": [\"34482201\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Causal ordering between WDFY4, ROS, p53, and ERK not dissected\",\n        \"No direct molecular target of WDFY4 identified\",\n        \"Single-lab study\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed WDFY4 restrains Th2 differentiation, broadening its immune role to allergic inflammation.\",\n      \"evidence\": \"Wdfy4-knockout mice with in vitro Th2 differentiation and OVA-induced asthma model with cytokine and histological readouts\",\n      \"pmids\": [\"34425575\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Mechanism by which WDFY4 limits Th2 polarization not defined\",\n        \"Cell-intrinsic versus extrinsic contribution not fully resolved\",\n        \"Single-lab study\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that WDFY4-dependent cross-presentation is not cDC1-restricted, establishing cDC2 as a sufficient cross-presenting cell for immune complex antigens.\",\n      \"evidence\": \"cDC1-deficient and Wdfy4-/- mice with in vivo immune complex/cell-associated antigen presentation and tumor rejection assays\",\n      \"pmids\": [\"39918736\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Molecular determinant of which antigens route through cDC2 versus cDC1 unknown\",\n        \"Whether WDFY4 acts identically in cDC1 and cDC2 not established at the mechanistic level\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed WDFY4 in an endothelial ferroptosis pathway through a direct interaction with LAPTM5 upstream of a CDC42/mTOR/4EBP1/SLC7A11 axis.\",\n      \"evidence\": \"Co-IP, immunofluorescence co-localization, endothelium-specific mouse models, and rescue experiments with LAPTM5 overexpression and CDC42 inhibition\",\n      \"pmids\": [\"40755163\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Downstream pathway partially inferred rather than fully demonstrated\",\n        \"Single-lab Co-IP without reciprocal validation\",\n        \"Relationship of this endothelial role to immune cross-presentation function unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The core biochemical mechanism by which WDFY4 enables antigen cross-presentation, and how its BEACH domain and diverse interaction partners coordinate this with its other immune and ferroptotic roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"No structural model or defined enzymatic/scaffolding activity for WDFY4\",\n        \"No identified molecular step in the cross-presentation pathway that WDFY4 executes\",\n        \"Unclear whether the distinct reported functions reflect one unifying mechanism or context-specific roles\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TLR3\", \"TLR4\", \"TLR9\", \"MDA5\", \"LAPTM5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}