{"gene":"HLA-F","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":1990,"finding":"HLA-F (HLA-5.4) encodes an intact class I protein with a shortened cytoplasmic tail compared to classical HLA molecules, and five of ten conserved antigen-binding residues are altered (three non-conservatively), suggesting a divergent peptide-binding function.","method":"DNA sequencing and protein sequence analysis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — original gene isolation and complete sequence determination establishing structural basis of the protein","pmids":["1688605"],"is_preprint":false},{"year":1990,"finding":"HLA-F protein, though intracellularly expressed after transfection into a B cell line, has a unique tissue distribution pattern (expressed in B cell lines, peripheral blood lymphocytes, absent from T cell lines and fibroblasts), and its transcript lacks exon 7 due to an altered acceptor splice site, resulting in a shorter cytoplasmic tail.","method":"Northern blot, cDNA cloning, transfection, Western blot","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 1 — direct molecular characterization with sequencing and functional transfection","pmids":["1707659"],"is_preprint":false},{"year":2000,"finding":"HLA-F heavy chain can be refolded with beta-2-microglobulin to form a stable complex; native HLA-F is predominantly intracellular and TAP-associated; HLA-F tetramers bind directly to ILT2 (LIR1) and ILT4 (LIR2) inhibitory receptors on monocytes and B cells, as confirmed by surface plasmon resonance.","method":"Recombinant protein refolding, monoclonal antibody production, immunoprecipitation, HLA-F tetramers, surface plasmon resonance, cell transfection","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — reconstituted protein complex, direct binding measured by SPR, and functional receptor identification","pmids":["11169396"],"is_preprint":false},{"year":2000,"finding":"HLA-F is expressed as a beta-2-microglobulin-associated 42-kDa empty heterodimer devoid of peptide; it is predominantly intracellular with immature oligosaccharide, associates with TAP and calreticulin, and IFN-gamma upregulates HLA-F mRNA and protein but does not induce cell surface expression.","method":"Immunoprecipitation, thermostability assays, endoglycosidase-H analysis, cell surface immunoprecipitation, Western blot","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal biochemical methods establishing peptide-free intracellular conformation and TAP/calreticulin association","pmids":["10605026"],"is_preprint":false},{"year":2000,"finding":"HLA-F transcription is regulated by NF-kappaB (via the kappaB1 site of enhancer A), responds to IFN-gamma through the ISRE, and is inducible by CIITA through the SXY regulatory module, distinguishing its regulation from HLA-G.","method":"Promoter sequence analysis, transactivation assays","journal":"Human immunology","confidence":"Medium","confidence_rationale":"Tier 2 — functional promoter dissection but single-lab study","pmids":["11137213"],"is_preprint":false},{"year":2003,"finding":"HLA-F surface expression on B lymphoblastoid and monocyte cell lines is partially independent from tapasin and completely independent from TAP, in contrast to classical MHC class I molecules; an Endo H-sensitive surface form is tapasin-independent while an Endo H-resistant form is tapasin-dependent.","method":"Immunoprecipitation, endoglycosidase-H treatment, flow cytometry, genetic cell lines deficient in TAP or tapasin","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods and use of defined genetic mutant cell lines","pmids":["14607927"],"is_preprint":false},{"year":2006,"finding":"HLA-F export from the endoplasmic reticulum depends entirely on its cytoplasmic tail: a C-terminal valine residue interacts with COPII for ER export, and an RxR motif binds 14-3-3 proteins for anterograde transport; classical class I molecules lacking their cytoplasmic tail are still surface expressed, while HLA-F is not.","method":"Cytoplasmic tail deletion and point mutation constructs, COPII interaction assay, 14-3-3 protein binding assay, surface expression analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis combined with identification of specific trafficking machinery interactions","pmids":["16709803"],"is_preprint":false},{"year":2010,"finding":"HLA-F is expressed intracellularly in all resting lymphocytes (B, T, NK, monocytes) without surface expression; upon activation of any lymphocyte subtype, surface HLA-F is induced. This surface upregulation occurs even in TAP- and tapasin-deficient individuals, though with altered kinetics. CD4+CD25+ Tregs do not upregulate surface HLA-F upon activation unlike CD4+CD25- T cells.","method":"Western blot, flow cytometry, activation of diverse lymphocyte subpopulations, analysis of genetically TAP/tapasin-deficient donor PBMCs","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — clean functional study with multiple cell types and genetic controls replicated across conditions","pmids":["20865824"],"is_preprint":false},{"year":2010,"finding":"HLA-F is expressed at the cell surface independently of bound peptide (as an open conformer), and physically interacts with MHC class I heavy chains only when the latter are in the open conformer (peptide-free) form; this interaction was demonstrated by co-immunoprecipitation, surface plasmon resonance, and tetramer/MHC-I HC colocalization on the cell surface.","method":"Direct peptide-binding analysis of native HLA-F, co-immunoprecipitation, surface plasmon resonance, tetramer staining, cell surface colocalization","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including SPR and co-IP, in a single rigorous study","pmids":["20483783"],"is_preprint":false},{"year":2013,"finding":"HLA-F expressed as a peptide-free open conformer physically and functionally interacts with the NK cell inhibitory receptor KIR3DL2 and activating receptor KIR2DS4; classical MHC-I open conformers also serve as ligands for these KIRs, defining a new category of KIR-MHC interactions centered on the inflammatory response.","method":"Surface plasmon resonance, biochemical pull-down from cell lines, functional NK cell assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 — SPR and biochemical pull-down with functional validation, single lab but multiple orthogonal methods","pmids":["24018270"],"is_preprint":false},{"year":2013,"finding":"HLA-F and MHC-I open conformers on activated lymphocytes and monocytes mediate a cross-presentation pathway for exogenous antigens that is independent of TAP and tapasin, sensitive to lysosomal enzyme inhibitors, and dependent on MHC-I allotype-specific epitope recognition for antigen uptake.","method":"In vitro cross-presentation assays, TAP/tapasin inhibition, lysosomal enzyme inhibitors, MHC-I allotype-specific blocking","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — functional pathway dissection with multiple pharmacological inhibitors, single lab","pmids":["23851683"],"is_preprint":false},{"year":2016,"finding":"HLA-F open conformers (peptide-free) are high-affinity ligands for the activating NK cell receptor KIR3DS1; activation of CD4+ T cells triggers surface expression of HLA-F, enabling KIR3DS1+ NK cell activation (degranulation, antiviral cytokine production); HIV-1 infection upregulates HLA-F mRNA but decreases KIR3DS1 binding, suggesting an immune evasion mechanism.","method":"Screening of 100 HLA class I proteins, biochemical binding assays, primary NK cell functional assays (degranulation, cytokine production), HIV-1 replication inhibition assay, flow cytometry","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1-2 — large-scale ligand screen, biochemical confirmation, and functional validation across multiple assays; independently replicated by subsequent studies","pmids":["27455421"],"is_preprint":false},{"year":2016,"finding":"KIR3DS1 but not KIR3DL1 physically binds HLA-F and other MHC-I open conformers, as measured by surface plasmon resonance; this was corroborated by biochemical pull-down and heterodimerization experiments with recombinant proteins; HLA-F ligation of KIR3DS1 triggers granule exocytosis in activated NK cells.","method":"Surface plasmon resonance, biochemical pull-down, recombinant protein heterodimerization, granule exocytosis assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal biophysical and biochemical methods with functional read-out","pmids":["27649529"],"is_preprint":false},{"year":2014,"finding":"HLA-F gene and protein expression are induced in JEV-infected human amniotic and endothelial cells via NFκB activation; shRNA knockdown of the p65 NFκB subunit blocks JEV-mediated HLA-F induction; TNF-alpha-driven HLA-F induction is also NFκB-dependent, and HLA-F enhancer A elements mediate NFκB-driven transactivation in reporter assays.","method":"RT-PCR, Western blot, shRNA knockdown of NFκB p65, luciferase reporter assay with HLA-F enhancer A elements, TNF-alpha stimulation","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 — shRNA knockdown and reporter assays in a single lab establishing NFκB as a transcriptional activator of HLA-F","pmids":["25461528"],"is_preprint":false},{"year":2018,"finding":"HLA-F is upregulated on HCV-infected cells; interactions between KIR3DS1 on NK cells and HLA-F on HCV-infected cells contribute to NK cell-mediated control of HCV replication in cell culture and humanized mouse liver models.","method":"Cell culture model of HCV infection, humanized mouse liver model, primary liver biopsy analysis, KIR3DS1/HLA-F blocking experiments","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple model systems (in vitro, in vivo humanized, ex vivo) with functional blocking, single lab","pmids":["30031767"],"is_preprint":false},{"year":2019,"finding":"HLA-F*01:01 presents peptides with non-canonical features: no defined N-terminal anchor, preferred length of 16 residues, and nearly all source proteins interact with HIV proteins; stable peptide-HLA-F complexes could be reconstituted from naturally presented peptides.","method":"Soluble HLA technology, LC-MS peptide elution and sequencing from stable pHLA-F*01:01 complexes","journal":"Immunogenetics","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical recovery and MS characterization of naturally presented peptides from reconstituted HLA-F complexes","pmids":["30941482"],"is_preprint":false},{"year":2019,"finding":"HLA-F allele-specific peptide selection differs completely among F*01:01, F*01:03, and F*01:04 despite polymorphisms outside peptide-binding pockets; all peptides are 8–24 amino acids, not N-terminally anchored, preferably C-terminally anchored by Lys; no proteomic overlap in peptide source proteins between allelic variants.","method":"Soluble HLA technology from K562 cells, LC-MS peptidome and proteome analysis, structural comparison of HLA-F allelic variants","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 — direct MS-based peptidome characterization from reconstituted allelic HLA-F complexes","pmids":["31717259"],"is_preprint":false},{"year":2020,"finding":"Peptide-bound HLA-F complexes do not bind soluble KIR3DS1; acid elution of peptides generates HLA-F open conformers that bind KIR3DS1; hemoglobin-derived peptides are dominant in HLA-F allelic peptide repertoires in CD4+ T cells post-HIV infection, and loading of hemoglobin peptide fractions onto HLA-F open conformers significantly diminishes KIR3DS1 receptor recognition, providing a molecular mechanism for HIV immune escape.","method":"Soluble HLA technology, acid elution, KIR3DS1 binding assay, MS proteome analysis of CD4+/HIV+ vs CD4+/HIV- cells, peptide binding and receptor recognition assay","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical reconstitution with functional receptor binding assays and MS-based peptide identification","pmids":["33126487"],"is_preprint":false},{"year":2019,"finding":"HLA-F on HIV-infected CD4+ T cells activates KIR3DS1+ NK cells (CCL4, IFN-gamma, CD107a), and blocking the HLA-F/KIR3DS1 interaction with KIR3DS1-Fc chimeric protein or anti-HLA-F antibody reduces the frequency of activated KIR3DS1+ NK cells.","method":"Co-culture of HIV-infected CD4+ T cells with primary NK cells, intracellular cytokine staining, degranulation assay, blocking with KIR3DS1-Fc chimera and anti-HLA-F mAb, exclusive gating strategies","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 — primary cell functional assays with specific blocking showing sufficiency of HLA-F/KIR3DS1 interaction","pmids":["31270222"],"is_preprint":false},{"year":2018,"finding":"HLA-F surface expression on the HLA-null cell line 721.221 activates primary KIR3DS1+ NK cells for CCL4, IFN-gamma, and CD107a secretion; blocking HLA-F on 721.221 cells with KIR3DS1-Fc chimeric protein or anti-HLA-F antibody reduces the frequency of activated KIR3DS1+ NK cells.","method":"Coculture of HLA-null 721.221 cells expressing HLA-F with primary NK cells, intracellular cytokine staining, degranulation assay, blocking experiments","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — use of HLA-null cell line isolates HLA-F contribution; confirmed by blocking, replicated in multiple conditions","pmids":["29743316"],"is_preprint":false},{"year":2020,"finding":"BK polyomavirus infection significantly increases surface expression of HLA-F on kidney tubular cells, enhancing KIR3DS1 binding and activating primary KIR3DS1+ NK cells, providing a mechanism for NK cell-mediated control of BKV infection.","method":"In vitro BKV infection model, flow cytometry of surface HLA-F, KIR3DS1 binding assay, primary NK cell activation assay, kidney biopsy analysis","journal":"Kidney international","confidence":"Medium","confidence_rationale":"Tier 2 — combined in vitro and ex vivo evidence with functional NK activation readout, single lab","pmids":["33359499"],"is_preprint":false},{"year":2021,"finding":"HLA-F promotes proliferation of glioma cells via HK2-dependent glycolysis: forced HLA-F expression increased HK2 protein stabilization and aerobic glycolysis phenotype; silencing HK2 reduced HLA-F-mediated glycolysis and cell proliferation.","method":"Forced HLA-F overexpression, HK2 shRNA knockdown, in vitro proliferation assays, glycolysis measurements, in vivo xenograft in immunodeficient Rag2 mice with anti-HLA-F antibody treatment","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function and gain-of-function with epistasis (HK2 silencing rescues phenotype), in vivo validation","pmids":["33867844"],"is_preprint":false},{"year":2025,"finding":"HLA-F regulates trophoblast proliferation via PKM2-dependent glycolysis: HLA-F overexpression promotes PKM2 protein expression and enzymatic activity, enhancing glycolysis; HLA-F binds the PKM promoter (shown by ChIP-seq) and reduces lactylation at PKM2 K305, increasing enzyme activity; PKM2 siRNA silencing abolishes HLA-F-mediated proliferation.","method":"ChIP-seq, 4D label-free quantitative proteomics, siRNA knockdown, CCK8/MTT/colony assay, Mini-PDX model, immunofluorescence, enzymatic activity assays","journal":"Molecular medicine","confidence":"Medium","confidence_rationale":"Tier 1-2 — ChIP-seq and proteomics with epistasis via PKM2 silencing; single lab","pmids":["40251569"],"is_preprint":false},{"year":2022,"finding":"HLA-F is mobilized to the surface of bronchial epithelial cells and platelets in an inflammatory/asthmatic context without changes at the transcriptional level, indicating surface expression is a ubiquitous post-transcriptional process in activated non-immune cells as well.","method":"Flow cytometry of surface HLA-F, RT-PCR for transcription, comparison of healthy vs. asthmatic donors, chemical activation experiments","journal":"HLA","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization data with functional inflammatory context, single lab","pmids":["35988034"],"is_preprint":false},{"year":2018,"finding":"An ancient G/A polymorphism at SNP rs2523393 creates a GATA2 binding site in a progesterone-responsive distal enhancer that loops to the HLA-F promoter, upregulating HLA-F expression in the endometrium during the window of implantation and in decidual stromal cells treated with progesterone.","method":"GTEx replication, reporter assay, chromatin looping analysis (3D genome), GATA2 binding site identification, progesterone treatment of decidual stromal cells","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — functional enhancer validated by looping and progesterone-responsive reporter, single lab","pmids":["30245028"],"is_preprint":false},{"year":2023,"finding":"HLA-F overexpression in Jar trophoblast cells promotes cell proliferation, invasion, and migration; in NK-92MI cells, HLA-F overexpression increases secretion of immunoregulation cytokines (CSF1, CCL22) and promotes adaptive NKG2C+ NK cell transformation.","method":"HLA-F lentiviral overexpression, CCK-8/proliferation assay, invasion/migration assay, cytokine measurement, flow cytometry of NK cell phenotypes","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 — gain-of-function with defined cellular phenotypes and cytokine readouts; single lab","pmids":["37854606"],"is_preprint":false}],"current_model":"HLA-F is a non-classical MHC class Ib molecule that is predominantly retained intracellularly as a peptide-free (open conformer) beta-2-microglobulin complex associated with TAP and calreticulin; upon lymphocyte or epithelial cell activation it is mobilized to the cell surface in a cytoplasmic tail-dependent manner (via COPII and 14-3-3 interactions), where it serves as a high-affinity ligand for the activating NK receptor KIR3DS1 and the inhibitory receptors ILT2/ILT4 (KIR3DL2, KIR2DS4), can present non-canonically long peptides without a defined N-terminal anchor, participates in TAP-independent MHC-I cross-presentation on activated cells, is transcriptionally induced by NF-kappaB, IFN-gamma/IRF1, and CIITA, and promotes trophoblast proliferation through PKM2-dependent glycolysis and glioma cell proliferation through HK2-dependent glycolysis."},"narrative":{"teleology":[{"year":1990,"claim":"Cloning of HLA-F revealed it encodes a structurally divergent MHC class I molecule with non-conservative substitutions in antigen-binding residues and a truncated cytoplasmic tail, establishing it as a non-classical class Ib gene with a predicted novel peptide-binding repertoire.","evidence":"DNA sequencing, protein sequence comparison, cDNA cloning and transfection in B cell lines","pmids":["1688605","1707659"],"confidence":"High","gaps":["No protein-level confirmation of peptide binding at this stage","Biological function completely unknown","Receptor partners unidentified"]},{"year":2000,"claim":"Biochemical characterization established that HLA-F is predominantly an intracellular, peptide-free beta-2-microglobulin heterodimer associated with TAP and calreticulin, and identified ILT2 and ILT4 as direct receptor partners, redefining HLA-F as an open-conformer ligand for inhibitory leukocyte receptors.","evidence":"Recombinant protein refolding, SPR binding, immunoprecipitation, tetramers, thermostability assays, endoglycosidase-H analysis","pmids":["11169396","10605026"],"confidence":"High","gaps":["Activating receptor partners unknown","Mechanism of ER retention vs. surface mobilization undefined","Whether HLA-F ever presents peptide in vivo unresolved"]},{"year":2000,"claim":"Dissection of HLA-F transcriptional regulation revealed NF-κB-, IFN-γ/ISRE-, and CIITA-dependent induction, placing HLA-F under both inflammatory and adaptive immune transcriptional control distinct from HLA-G.","evidence":"Promoter sequence analysis and transactivation assays","pmids":["11137213"],"confidence":"Medium","gaps":["In vivo confirmation of each transcription factor's contribution lacking","Post-transcriptional regulation not addressed","Cell-type specificity of transcriptional control unexplored"]},{"year":2003,"claim":"Demonstration that HLA-F surface expression is TAP-independent and partially tapasin-independent resolved the question of how a peptide-free molecule reaches the surface, distinguishing HLA-F trafficking from classical MHC-I.","evidence":"Flow cytometry and endoglycosidase-H treatment in TAP- and tapasin-deficient cell lines","pmids":["14607927"],"confidence":"High","gaps":["Positive trafficking signals not yet identified","Whether surface HLA-F carries any peptide cargo unresolved"]},{"year":2006,"claim":"Identification of cytoplasmic tail motifs (C-terminal valine for COPII, RxR for 14-3-3) as essential for HLA-F ER export provided a molecular explanation for its predominantly intracellular retention and activation-dependent surface mobilization.","evidence":"Tail deletion/point mutants, COPII and 14-3-3 interaction assays, surface expression analysis","pmids":["16709803"],"confidence":"High","gaps":["Upstream signal linking cell activation to tail-dependent export unknown","Whether 14-3-3 binding is phosphorylation-regulated not tested"]},{"year":2010,"claim":"Systematic analysis across lymphocyte subsets showed that all resting lymphocytes store intracellular HLA-F and mobilize it to the surface upon activation — except regulatory T cells — establishing HLA-F surface display as a universal activation marker and defining its peptide-free open conformer as the predominant surface species that interacts with classical MHC-I open conformers.","evidence":"Flow cytometry and Western blot across B, T, NK, monocyte subsets; TAP/tapasin-deficient donor PBMCs; co-IP, SPR, and tetramer colocalization","pmids":["20865824","20483783"],"confidence":"High","gaps":["Functional consequence of HLA-F–MHC-I open conformer interaction on signaling unknown","Treg-specific suppression mechanism undefined"]},{"year":2013,"claim":"Identification of KIR3DL2 and KIR2DS4 as additional receptors for HLA-F open conformers, together with a TAP-independent cross-presentation pathway mediated by HLA-F and MHC-I open conformers, expanded the functional repertoire from inhibitory signaling to both activating and antigen-presenting roles.","evidence":"SPR, biochemical pull-down, functional NK assays; cross-presentation assays with lysosomal inhibitors and MHC-I allotype blocking","pmids":["24018270","23851683"],"confidence":"High","gaps":["Structural basis of KIR recognition of open conformers undefined","Cross-presentation pathway's in vivo relevance unestablished","Whether HLA-F itself cross-presents peptide or serves as co-receptor unclear"]},{"year":2016,"claim":"A comprehensive screen identified HLA-F open conformers as high-affinity ligands for the activating receptor KIR3DS1, establishing the HLA-F/KIR3DS1 axis as a major activating pathway for NK cell degranulation and antiviral cytokine production.","evidence":"Screening 100 HLA class I proteins, SPR, primary NK cell functional assays, HIV replication inhibition, HLA-null cell line blocking experiments","pmids":["27455421","27649529","29743316"],"confidence":"High","gaps":["Crystal structure of HLA-F/KIR3DS1 complex not solved","Whether KIR3DS1 distinguishes among HLA-F alleles unknown"]},{"year":2019,"claim":"Mass spectrometry-based peptidome characterization of HLA-F revealed presentation of unconventionally long peptides (8–24 residues) without N-terminal anchoring but with C-terminal lysine preference, and showed complete allele-specific peptide repertoire divergence, defining a non-canonical antigen presentation mode.","evidence":"Soluble HLA technology, LC-MS peptidome from reconstituted HLA-F*01:01, *01:03, *01:04 complexes","pmids":["30941482","31717259"],"confidence":"High","gaps":["In vivo T cell recognition of HLA-F-presented peptides undemonstrated","Structural basis of long-peptide accommodation unknown","Whether peptide-loaded HLA-F engages any receptor besides KIRs untested"]},{"year":2020,"claim":"Biochemical reconstitution showed that peptide loading onto HLA-F open conformers abolishes KIR3DS1 binding, establishing a molecular switch: peptide-free HLA-F activates NK cells via KIR3DS1 while peptide-loaded HLA-F does not, and HIV-driven enrichment of hemoglobin-derived peptides on HLA-F provides a viral immune evasion mechanism.","evidence":"Acid elution to generate open conformers, KIR3DS1 binding assay, MS proteomics of HIV-infected CD4+ T cells, peptide fraction loading and receptor recognition assay","pmids":["33126487"],"confidence":"High","gaps":["How HIV promotes hemoglobin peptide loading specifically onto HLA-F is unknown","Whether other viruses exploit the same evasion mechanism untested"]},{"year":2018,"claim":"Discovery of a progesterone-responsive distal enhancer with an ancient polymorphism creating a GATA2 binding site that loops to the HLA-F promoter revealed tissue-specific endometrial regulation, connecting HLA-F to reproductive immunology and implantation.","evidence":"GTEx replication, reporter assay, chromatin looping analysis, progesterone treatment of decidual stromal cells","pmids":["30245028"],"confidence":"Medium","gaps":["Functional consequence of enhanced HLA-F expression during implantation for NK cell tolerance unknown","Whether this enhancer operates in trophoblasts themselves untested"]},{"year":2021,"claim":"An unexpected non-immune function was established: HLA-F promotes glioma and trophoblast cell proliferation via metabolic reprogramming through stabilization of glycolytic enzymes HK2 and PKM2 respectively, with epistatic rescue upon enzyme silencing.","evidence":"Overexpression/knockdown, glycolysis measurements, xenograft models, ChIP-seq for PKM promoter binding, siRNA epistasis","pmids":["33867844","40251569"],"confidence":"Medium","gaps":["How an MHC-I molecule directly regulates glycolytic enzyme expression/stability is mechanistically unexplained","Whether metabolic effects occur in immune cell contexts untested","Independent replication needed"]},{"year":null,"claim":"Key unresolved questions include the structural basis of HLA-F open conformer recognition by KIR3DS1, the mechanism by which HLA-F regulates glycolytic enzymes, whether peptide-loaded HLA-F engages adaptive T cell receptors in vivo, and how the switch between open conformer and peptide-loaded states is regulated during inflammation.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure of HLA-F/KIR3DS1 complex","No TCR recognition of HLA-F-peptide complexes demonstrated","Molecular mechanism linking HLA-F to HK2/PKM2 stabilization uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[2,9,11,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,17,18,19]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3,6]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,8,11,23]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,3,7]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,9,10,11,12,18,19]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[21,22]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[24,25]}],"complexes":["MHC class I peptide-loading complex (TAP/calreticulin)"],"partners":["KIR3DS1","ILT2","ILT4","KIR3DL2","KIR2DS4","B2M","HK2","PKM2"],"other_free_text":[]},"mechanistic_narrative":"HLA-F is a non-classical MHC class Ib molecule that functions as a versatile immune regulator, serving both as a ligand for innate immune receptors on NK cells and as an unconventional peptide presenter on activated cells. Structurally, HLA-F forms a peptide-free open conformer associated with beta-2-microglobulin, TAP, and calreticulin in the endoplasmic reticulum; its surface mobilization upon cellular activation depends on cytoplasmic tail motifs that engage COPII and 14-3-3 trafficking machinery rather than on conventional peptide loading [PMID:10605026, PMID:16709803, PMID:20865824]. At the cell surface, HLA-F open conformers serve as high-affinity ligands for the activating NK receptor KIR3DS1 and the inhibitory receptors ILT2, ILT4, KIR3DL2, and KIR2DS4, thereby triggering NK cell degranulation and cytokine production during viral infections including HIV, HCV, and BKV [PMID:27455421, PMID:11169396, PMID:24018270, PMID:30031767]. When peptide-loaded, HLA-F presents non-canonically long peptides (8–24 residues) lacking a classical N-terminal anchor, with allele-specific repertoires and C-terminal lysine preference; peptide occupancy abolishes KIR3DS1 recognition, providing a molecular switch between activating and tolerogenic signaling [PMID:30941482, PMID:31717259, PMID:33126487]."},"prefetch_data":{"uniprot":{"accession":"P30511","full_name":"HLA class I histocompatibility antigen, alpha chain F","aliases":["CDA12","HLA F antigen","Leukocyte antigen F","MHC class I antigen F"],"length_aa":346,"mass_kda":39.1,"function":"Non-classical major histocompatibility class Ib molecule postulated to play a role in immune surveillance, immune tolerance and inflammation. Functions in two forms, as a heterotrimeric complex with B2M/beta-2 microglobulin and a peptide (peptide-bound HLA-F-B2M) and as an open conformer (OC) devoid of peptide and B2M (peptide-free OC). In complex with B2M, presents non-canonical self-peptides carrying post-translational modifications, particularly phosphorylated self-peptides. Peptide-bound HLA-F-B2M acts as a ligand for LILRB1 inhibitory receptor, a major player in maternal-fetal tolerance. Peptide-free OC acts as a ligand for KIR3DS1 and KIR3DL2 receptors (PubMed:28636952). Upon interaction with activating KIR3DS1 receptor on NK cells, triggers NK cell degranulation and anti-viral cytokine production (PubMed:27455421). Through interaction with KIR3DL2 receptor, inhibits NK and T cell effector functions (PubMed:24018270). May interact with other MHC class I OCs to cross-present exogenous viral, tumor or minor histompatibility antigens to cytotoxic CD8+ T cells, triggering effector and memory responses (PubMed:23851683). May play a role in inflammatory responses in the peripheral nervous system. Through interaction with KIR3DL2, may protect motor neurons from astrocyte-induced toxicity (PubMed:26928464)","subcellular_location":"Cell membrane; Early endosome membrane; Lysosome membrane","url":"https://www.uniprot.org/uniprotkb/P30511/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HLA-F","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HLA-F","total_profiled":1310},"omim":[{"mim_id":"620778","title":"KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTOR, THREE DOMAINS, SHORT CYTOPLASMIC TAIL, 1; KIR3DS1","url":"https://www.omim.org/entry/620778"},{"mim_id":"615797","title":"HLA COMPLEX GROUP 9, NONCODING; HCG9","url":"https://www.omim.org/entry/615797"},{"mim_id":"615714","title":"POLR1H ANTISENSE, PSEUDOGENE; POLR1HASP","url":"https://www.omim.org/entry/615714"},{"mim_id":"613609","title":"HOMEOSTATIC IRON REGULATOR; HFE","url":"https://www.omim.org/entry/613609"},{"mim_id":"607525","title":"POLYMERASE I, RNA, SUBUNIT H; POLR1H","url":"https://www.omim.org/entry/607525"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":172.8}],"url":"https://www.proteinatlas.org/search/HLA-F"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P30511","domains":[{"cath_id":"3.30.500.10","chopping":"23-200","consensus_level":"high","plddt":97.0535,"start":23,"end":200},{"cath_id":"2.60.40.10","chopping":"206-293","consensus_level":"high","plddt":96.4612,"start":206,"end":293}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P30511","model_url":"https://alphafold.ebi.ac.uk/files/AF-P30511-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P30511-F1-predicted_aligned_error_v6.png","plddt_mean":89.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HLA-F","jax_strain_url":"https://www.jax.org/strain/search?query=HLA-F"},"sequence":{"accession":"P30511","fasta_url":"https://rest.uniprot.org/uniprotkb/P30511.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P30511/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P30511"}},"corpus_meta":[{"pmid":"2249951","id":"PMC_2249951","title":"Differential expression of HLA-E, HLA-F, and HLA-G transcripts in human tissue.","date":"1990","source":"Human immunology","url":"https://pubmed.ncbi.nlm.nih.gov/2249951","citation_count":225,"is_preprint":false},{"pmid":"1688605","id":"PMC_1688605","title":"Human leukocyte antigen F (HLA-F). 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genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8566959","citation_count":4,"is_preprint":false},{"pmid":"33184728","id":"PMC_33184728","title":"Evolution of HLA-F and its orthologues in primate species: a complex tale of conservation, diversification and inactivation.","date":"2020","source":"Immunogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/33184728","citation_count":4,"is_preprint":false},{"pmid":"34934358","id":"PMC_34934358","title":"LncRNA HLA-F-AS1 Enhances the Migration, Invasion and Apoptosis of Glioblastoma Cells by Targeting lncRNA MEG3.","date":"2021","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/34934358","citation_count":3,"is_preprint":false},{"pmid":"35447336","id":"PMC_35447336","title":"Epigenome-Wide DNA Methylation Profiling of Normal Mucosa Reveals HLA-F Hypermethylation as a Biomarker Candidate for Serrated Polyposis Syndrome.","date":"2022","source":"The Journal of molecular diagnostics : JMD","url":"https://pubmed.ncbi.nlm.nih.gov/35447336","citation_count":3,"is_preprint":false},{"pmid":"35935961","id":"PMC_35935961","title":"Sequence Variations Within HLA-G and HLA-F Genomic Segments at the Human Leukocyte Antigen Telomeric End Associated With Acute Graft-Versus-Host Disease in Unrelated Bone Marrow Transplantation.","date":"2022","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35935961","citation_count":3,"is_preprint":false},{"pmid":"38568442","id":"PMC_38568442","title":"Potentials as biomarker and therapeutic target of upregulated long non-coding RNA HLA-F antisense RNA 1 in hepatitis B virus-associated hepatocellular carcinoma.","date":"2024","source":"Virus genes","url":"https://pubmed.ncbi.nlm.nih.gov/38568442","citation_count":3,"is_preprint":false},{"pmid":"33420581","id":"PMC_33420581","title":"DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B.","date":"2021","source":"Chinese journal of integrative medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33420581","citation_count":2,"is_preprint":false},{"pmid":"33781967","id":"PMC_33781967","title":"Human leukocyte antigen (HLA)-F and -G gene polymorphisms and haplotypes are associated with malaria susceptibility in the Beninese Toffin children.","date":"2021","source":"Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/33781967","citation_count":2,"is_preprint":false},{"pmid":"38390869","id":"PMC_38390869","title":"Diversity in the HLA-I Recognition of HLA-F Monoclonal Antibodies: HLA-F or HLA-Ib Monospecific, HLA-E or HLA-G Bispecific Antibodies with or without HLA-Ia Reactivity.","date":"2024","source":"Antibodies (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/38390869","citation_count":2,"is_preprint":false},{"pmid":"39405828","id":"PMC_39405828","title":"Effect modification between HLA-F and CD56 markers reveals differences in survival for triple-negative breast cancer patients.","date":"2024","source":"Human immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39405828","citation_count":1,"is_preprint":false},{"pmid":"40184242","id":"PMC_40184242","title":"Association of HLA-Ib (HLA-G, HLA-E and HLA-F) with spontaneous HBV clearance.","date":"2025","source":"Infectious diseases (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/40184242","citation_count":1,"is_preprint":false},{"pmid":"38073430","id":"PMC_38073430","title":"The novel HLA-F*01:16 and HLA-F*01:17 alleles identified in hematopoietic cell 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genetics","url":"https://pubmed.ncbi.nlm.nih.gov/41321564","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.28.615513","title":"Exploring the Correlation Between UVB Sensitivity and SLE Activity: Insights into UVB-Driven Pathogenesis in Lupus Erythematosus","date":"2024-09-30","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.28.615513","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":45634,"output_tokens":6096,"usd":0.114171},"stage2":{"model":"claude-opus-4-6","input_tokens":9717,"output_tokens":3391,"usd":0.20004},"total_usd":0.314211,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1990,\n      \"finding\": \"HLA-F (HLA-5.4) encodes an intact class I protein with a shortened cytoplasmic tail compared to classical HLA molecules, and five of ten conserved antigen-binding residues are altered (three non-conservatively), suggesting a divergent peptide-binding function.\",\n      \"method\": \"DNA sequencing and protein sequence analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original gene isolation and complete sequence determination establishing structural basis of the protein\",\n      \"pmids\": [\"1688605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"HLA-F protein, though intracellularly expressed after transfection into a B cell line, has a unique tissue distribution pattern (expressed in B cell lines, peripheral blood lymphocytes, absent from T cell lines and fibroblasts), and its transcript lacks exon 7 due to an altered acceptor splice site, resulting in a shorter cytoplasmic tail.\",\n      \"method\": \"Northern blot, cDNA cloning, transfection, Western blot\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct molecular characterization with sequencing and functional transfection\",\n      \"pmids\": [\"1707659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HLA-F heavy chain can be refolded with beta-2-microglobulin to form a stable complex; native HLA-F is predominantly intracellular and TAP-associated; HLA-F tetramers bind directly to ILT2 (LIR1) and ILT4 (LIR2) inhibitory receptors on monocytes and B cells, as confirmed by surface plasmon resonance.\",\n      \"method\": \"Recombinant protein refolding, monoclonal antibody production, immunoprecipitation, HLA-F tetramers, surface plasmon resonance, cell transfection\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted protein complex, direct binding measured by SPR, and functional receptor identification\",\n      \"pmids\": [\"11169396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HLA-F is expressed as a beta-2-microglobulin-associated 42-kDa empty heterodimer devoid of peptide; it is predominantly intracellular with immature oligosaccharide, associates with TAP and calreticulin, and IFN-gamma upregulates HLA-F mRNA and protein but does not induce cell surface expression.\",\n      \"method\": \"Immunoprecipitation, thermostability assays, endoglycosidase-H analysis, cell surface immunoprecipitation, Western blot\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal biochemical methods establishing peptide-free intracellular conformation and TAP/calreticulin association\",\n      \"pmids\": [\"10605026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HLA-F transcription is regulated by NF-kappaB (via the kappaB1 site of enhancer A), responds to IFN-gamma through the ISRE, and is inducible by CIITA through the SXY regulatory module, distinguishing its regulation from HLA-G.\",\n      \"method\": \"Promoter sequence analysis, transactivation assays\",\n      \"journal\": \"Human immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional promoter dissection but single-lab study\",\n      \"pmids\": [\"11137213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"HLA-F surface expression on B lymphoblastoid and monocyte cell lines is partially independent from tapasin and completely independent from TAP, in contrast to classical MHC class I molecules; an Endo H-sensitive surface form is tapasin-independent while an Endo H-resistant form is tapasin-dependent.\",\n      \"method\": \"Immunoprecipitation, endoglycosidase-H treatment, flow cytometry, genetic cell lines deficient in TAP or tapasin\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods and use of defined genetic mutant cell lines\",\n      \"pmids\": [\"14607927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"HLA-F export from the endoplasmic reticulum depends entirely on its cytoplasmic tail: a C-terminal valine residue interacts with COPII for ER export, and an RxR motif binds 14-3-3 proteins for anterograde transport; classical class I molecules lacking their cytoplasmic tail are still surface expressed, while HLA-F is not.\",\n      \"method\": \"Cytoplasmic tail deletion and point mutation constructs, COPII interaction assay, 14-3-3 protein binding assay, surface expression analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis combined with identification of specific trafficking machinery interactions\",\n      \"pmids\": [\"16709803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HLA-F is expressed intracellularly in all resting lymphocytes (B, T, NK, monocytes) without surface expression; upon activation of any lymphocyte subtype, surface HLA-F is induced. This surface upregulation occurs even in TAP- and tapasin-deficient individuals, though with altered kinetics. CD4+CD25+ Tregs do not upregulate surface HLA-F upon activation unlike CD4+CD25- T cells.\",\n      \"method\": \"Western blot, flow cytometry, activation of diverse lymphocyte subpopulations, analysis of genetically TAP/tapasin-deficient donor PBMCs\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean functional study with multiple cell types and genetic controls replicated across conditions\",\n      \"pmids\": [\"20865824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HLA-F is expressed at the cell surface independently of bound peptide (as an open conformer), and physically interacts with MHC class I heavy chains only when the latter are in the open conformer (peptide-free) form; this interaction was demonstrated by co-immunoprecipitation, surface plasmon resonance, and tetramer/MHC-I HC colocalization on the cell surface.\",\n      \"method\": \"Direct peptide-binding analysis of native HLA-F, co-immunoprecipitation, surface plasmon resonance, tetramer staining, cell surface colocalization\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including SPR and co-IP, in a single rigorous study\",\n      \"pmids\": [\"20483783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HLA-F expressed as a peptide-free open conformer physically and functionally interacts with the NK cell inhibitory receptor KIR3DL2 and activating receptor KIR2DS4; classical MHC-I open conformers also serve as ligands for these KIRs, defining a new category of KIR-MHC interactions centered on the inflammatory response.\",\n      \"method\": \"Surface plasmon resonance, biochemical pull-down from cell lines, functional NK cell assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — SPR and biochemical pull-down with functional validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24018270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HLA-F and MHC-I open conformers on activated lymphocytes and monocytes mediate a cross-presentation pathway for exogenous antigens that is independent of TAP and tapasin, sensitive to lysosomal enzyme inhibitors, and dependent on MHC-I allotype-specific epitope recognition for antigen uptake.\",\n      \"method\": \"In vitro cross-presentation assays, TAP/tapasin inhibition, lysosomal enzyme inhibitors, MHC-I allotype-specific blocking\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional pathway dissection with multiple pharmacological inhibitors, single lab\",\n      \"pmids\": [\"23851683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HLA-F open conformers (peptide-free) are high-affinity ligands for the activating NK cell receptor KIR3DS1; activation of CD4+ T cells triggers surface expression of HLA-F, enabling KIR3DS1+ NK cell activation (degranulation, antiviral cytokine production); HIV-1 infection upregulates HLA-F mRNA but decreases KIR3DS1 binding, suggesting an immune evasion mechanism.\",\n      \"method\": \"Screening of 100 HLA class I proteins, biochemical binding assays, primary NK cell functional assays (degranulation, cytokine production), HIV-1 replication inhibition assay, flow cytometry\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — large-scale ligand screen, biochemical confirmation, and functional validation across multiple assays; independently replicated by subsequent studies\",\n      \"pmids\": [\"27455421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KIR3DS1 but not KIR3DL1 physically binds HLA-F and other MHC-I open conformers, as measured by surface plasmon resonance; this was corroborated by biochemical pull-down and heterodimerization experiments with recombinant proteins; HLA-F ligation of KIR3DS1 triggers granule exocytosis in activated NK cells.\",\n      \"method\": \"Surface plasmon resonance, biochemical pull-down, recombinant protein heterodimerization, granule exocytosis assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal biophysical and biochemical methods with functional read-out\",\n      \"pmids\": [\"27649529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HLA-F gene and protein expression are induced in JEV-infected human amniotic and endothelial cells via NFκB activation; shRNA knockdown of the p65 NFκB subunit blocks JEV-mediated HLA-F induction; TNF-alpha-driven HLA-F induction is also NFκB-dependent, and HLA-F enhancer A elements mediate NFκB-driven transactivation in reporter assays.\",\n      \"method\": \"RT-PCR, Western blot, shRNA knockdown of NFκB p65, luciferase reporter assay with HLA-F enhancer A elements, TNF-alpha stimulation\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — shRNA knockdown and reporter assays in a single lab establishing NFκB as a transcriptional activator of HLA-F\",\n      \"pmids\": [\"25461528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HLA-F is upregulated on HCV-infected cells; interactions between KIR3DS1 on NK cells and HLA-F on HCV-infected cells contribute to NK cell-mediated control of HCV replication in cell culture and humanized mouse liver models.\",\n      \"method\": \"Cell culture model of HCV infection, humanized mouse liver model, primary liver biopsy analysis, KIR3DS1/HLA-F blocking experiments\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple model systems (in vitro, in vivo humanized, ex vivo) with functional blocking, single lab\",\n      \"pmids\": [\"30031767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HLA-F*01:01 presents peptides with non-canonical features: no defined N-terminal anchor, preferred length of 16 residues, and nearly all source proteins interact with HIV proteins; stable peptide-HLA-F complexes could be reconstituted from naturally presented peptides.\",\n      \"method\": \"Soluble HLA technology, LC-MS peptide elution and sequencing from stable pHLA-F*01:01 complexes\",\n      \"journal\": \"Immunogenetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical recovery and MS characterization of naturally presented peptides from reconstituted HLA-F complexes\",\n      \"pmids\": [\"30941482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HLA-F allele-specific peptide selection differs completely among F*01:01, F*01:03, and F*01:04 despite polymorphisms outside peptide-binding pockets; all peptides are 8–24 amino acids, not N-terminally anchored, preferably C-terminally anchored by Lys; no proteomic overlap in peptide source proteins between allelic variants.\",\n      \"method\": \"Soluble HLA technology from K562 cells, LC-MS peptidome and proteome analysis, structural comparison of HLA-F allelic variants\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct MS-based peptidome characterization from reconstituted allelic HLA-F complexes\",\n      \"pmids\": [\"31717259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Peptide-bound HLA-F complexes do not bind soluble KIR3DS1; acid elution of peptides generates HLA-F open conformers that bind KIR3DS1; hemoglobin-derived peptides are dominant in HLA-F allelic peptide repertoires in CD4+ T cells post-HIV infection, and loading of hemoglobin peptide fractions onto HLA-F open conformers significantly diminishes KIR3DS1 receptor recognition, providing a molecular mechanism for HIV immune escape.\",\n      \"method\": \"Soluble HLA technology, acid elution, KIR3DS1 binding assay, MS proteome analysis of CD4+/HIV+ vs CD4+/HIV- cells, peptide binding and receptor recognition assay\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical reconstitution with functional receptor binding assays and MS-based peptide identification\",\n      \"pmids\": [\"33126487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HLA-F on HIV-infected CD4+ T cells activates KIR3DS1+ NK cells (CCL4, IFN-gamma, CD107a), and blocking the HLA-F/KIR3DS1 interaction with KIR3DS1-Fc chimeric protein or anti-HLA-F antibody reduces the frequency of activated KIR3DS1+ NK cells.\",\n      \"method\": \"Co-culture of HIV-infected CD4+ T cells with primary NK cells, intracellular cytokine staining, degranulation assay, blocking with KIR3DS1-Fc chimera and anti-HLA-F mAb, exclusive gating strategies\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — primary cell functional assays with specific blocking showing sufficiency of HLA-F/KIR3DS1 interaction\",\n      \"pmids\": [\"31270222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HLA-F surface expression on the HLA-null cell line 721.221 activates primary KIR3DS1+ NK cells for CCL4, IFN-gamma, and CD107a secretion; blocking HLA-F on 721.221 cells with KIR3DS1-Fc chimeric protein or anti-HLA-F antibody reduces the frequency of activated KIR3DS1+ NK cells.\",\n      \"method\": \"Coculture of HLA-null 721.221 cells expressing HLA-F with primary NK cells, intracellular cytokine staining, degranulation assay, blocking experiments\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — use of HLA-null cell line isolates HLA-F contribution; confirmed by blocking, replicated in multiple conditions\",\n      \"pmids\": [\"29743316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BK polyomavirus infection significantly increases surface expression of HLA-F on kidney tubular cells, enhancing KIR3DS1 binding and activating primary KIR3DS1+ NK cells, providing a mechanism for NK cell-mediated control of BKV infection.\",\n      \"method\": \"In vitro BKV infection model, flow cytometry of surface HLA-F, KIR3DS1 binding assay, primary NK cell activation assay, kidney biopsy analysis\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — combined in vitro and ex vivo evidence with functional NK activation readout, single lab\",\n      \"pmids\": [\"33359499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HLA-F promotes proliferation of glioma cells via HK2-dependent glycolysis: forced HLA-F expression increased HK2 protein stabilization and aerobic glycolysis phenotype; silencing HK2 reduced HLA-F-mediated glycolysis and cell proliferation.\",\n      \"method\": \"Forced HLA-F overexpression, HK2 shRNA knockdown, in vitro proliferation assays, glycolysis measurements, in vivo xenograft in immunodeficient Rag2 mice with anti-HLA-F antibody treatment\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function and gain-of-function with epistasis (HK2 silencing rescues phenotype), in vivo validation\",\n      \"pmids\": [\"33867844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HLA-F regulates trophoblast proliferation via PKM2-dependent glycolysis: HLA-F overexpression promotes PKM2 protein expression and enzymatic activity, enhancing glycolysis; HLA-F binds the PKM promoter (shown by ChIP-seq) and reduces lactylation at PKM2 K305, increasing enzyme activity; PKM2 siRNA silencing abolishes HLA-F-mediated proliferation.\",\n      \"method\": \"ChIP-seq, 4D label-free quantitative proteomics, siRNA knockdown, CCK8/MTT/colony assay, Mini-PDX model, immunofluorescence, enzymatic activity assays\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP-seq and proteomics with epistasis via PKM2 silencing; single lab\",\n      \"pmids\": [\"40251569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HLA-F is mobilized to the surface of bronchial epithelial cells and platelets in an inflammatory/asthmatic context without changes at the transcriptional level, indicating surface expression is a ubiquitous post-transcriptional process in activated non-immune cells as well.\",\n      \"method\": \"Flow cytometry of surface HLA-F, RT-PCR for transcription, comparison of healthy vs. asthmatic donors, chemical activation experiments\",\n      \"journal\": \"HLA\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization data with functional inflammatory context, single lab\",\n      \"pmids\": [\"35988034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"An ancient G/A polymorphism at SNP rs2523393 creates a GATA2 binding site in a progesterone-responsive distal enhancer that loops to the HLA-F promoter, upregulating HLA-F expression in the endometrium during the window of implantation and in decidual stromal cells treated with progesterone.\",\n      \"method\": \"GTEx replication, reporter assay, chromatin looping analysis (3D genome), GATA2 binding site identification, progesterone treatment of decidual stromal cells\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional enhancer validated by looping and progesterone-responsive reporter, single lab\",\n      \"pmids\": [\"30245028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HLA-F overexpression in Jar trophoblast cells promotes cell proliferation, invasion, and migration; in NK-92MI cells, HLA-F overexpression increases secretion of immunoregulation cytokines (CSF1, CCL22) and promotes adaptive NKG2C+ NK cell transformation.\",\n      \"method\": \"HLA-F lentiviral overexpression, CCK-8/proliferation assay, invasion/migration assay, cytokine measurement, flow cytometry of NK cell phenotypes\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function with defined cellular phenotypes and cytokine readouts; single lab\",\n      \"pmids\": [\"37854606\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HLA-F is a non-classical MHC class Ib molecule that is predominantly retained intracellularly as a peptide-free (open conformer) beta-2-microglobulin complex associated with TAP and calreticulin; upon lymphocyte or epithelial cell activation it is mobilized to the cell surface in a cytoplasmic tail-dependent manner (via COPII and 14-3-3 interactions), where it serves as a high-affinity ligand for the activating NK receptor KIR3DS1 and the inhibitory receptors ILT2/ILT4 (KIR3DL2, KIR2DS4), can present non-canonically long peptides without a defined N-terminal anchor, participates in TAP-independent MHC-I cross-presentation on activated cells, is transcriptionally induced by NF-kappaB, IFN-gamma/IRF1, and CIITA, and promotes trophoblast proliferation through PKM2-dependent glycolysis and glioma cell proliferation through HK2-dependent glycolysis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HLA-F is a non-classical MHC class Ib molecule that functions as a versatile immune regulator, serving both as a ligand for innate immune receptors on NK cells and as an unconventional peptide presenter on activated cells. Structurally, HLA-F forms a peptide-free open conformer associated with beta-2-microglobulin, TAP, and calreticulin in the endoplasmic reticulum; its surface mobilization upon cellular activation depends on cytoplasmic tail motifs that engage COPII and 14-3-3 trafficking machinery rather than on conventional peptide loading [PMID:10605026, PMID:16709803, PMID:20865824]. At the cell surface, HLA-F open conformers serve as high-affinity ligands for the activating NK receptor KIR3DS1 and the inhibitory receptors ILT2, ILT4, KIR3DL2, and KIR2DS4, thereby triggering NK cell degranulation and cytokine production during viral infections including HIV, HCV, and BKV [PMID:27455421, PMID:11169396, PMID:24018270, PMID:30031767]. When peptide-loaded, HLA-F presents non-canonically long peptides (8–24 residues) lacking a classical N-terminal anchor, with allele-specific repertoires and C-terminal lysine preference; peptide occupancy abolishes KIR3DS1 recognition, providing a molecular switch between activating and tolerogenic signaling [PMID:30941482, PMID:31717259, PMID:33126487].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Cloning of HLA-F revealed it encodes a structurally divergent MHC class I molecule with non-conservative substitutions in antigen-binding residues and a truncated cytoplasmic tail, establishing it as a non-classical class Ib gene with a predicted novel peptide-binding repertoire.\",\n      \"evidence\": \"DNA sequencing, protein sequence comparison, cDNA cloning and transfection in B cell lines\",\n      \"pmids\": [\"1688605\", \"1707659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No protein-level confirmation of peptide binding at this stage\", \"Biological function completely unknown\", \"Receptor partners unidentified\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Biochemical characterization established that HLA-F is predominantly an intracellular, peptide-free beta-2-microglobulin heterodimer associated with TAP and calreticulin, and identified ILT2 and ILT4 as direct receptor partners, redefining HLA-F as an open-conformer ligand for inhibitory leukocyte receptors.\",\n      \"evidence\": \"Recombinant protein refolding, SPR binding, immunoprecipitation, tetramers, thermostability assays, endoglycosidase-H analysis\",\n      \"pmids\": [\"11169396\", \"10605026\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Activating receptor partners unknown\", \"Mechanism of ER retention vs. surface mobilization undefined\", \"Whether HLA-F ever presents peptide in vivo unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Dissection of HLA-F transcriptional regulation revealed NF-κB-, IFN-γ/ISRE-, and CIITA-dependent induction, placing HLA-F under both inflammatory and adaptive immune transcriptional control distinct from HLA-G.\",\n      \"evidence\": \"Promoter sequence analysis and transactivation assays\",\n      \"pmids\": [\"11137213\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo confirmation of each transcription factor's contribution lacking\", \"Post-transcriptional regulation not addressed\", \"Cell-type specificity of transcriptional control unexplored\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstration that HLA-F surface expression is TAP-independent and partially tapasin-independent resolved the question of how a peptide-free molecule reaches the surface, distinguishing HLA-F trafficking from classical MHC-I.\",\n      \"evidence\": \"Flow cytometry and endoglycosidase-H treatment in TAP- and tapasin-deficient cell lines\",\n      \"pmids\": [\"14607927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Positive trafficking signals not yet identified\", \"Whether surface HLA-F carries any peptide cargo unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identification of cytoplasmic tail motifs (C-terminal valine for COPII, RxR for 14-3-3) as essential for HLA-F ER export provided a molecular explanation for its predominantly intracellular retention and activation-dependent surface mobilization.\",\n      \"evidence\": \"Tail deletion/point mutants, COPII and 14-3-3 interaction assays, surface expression analysis\",\n      \"pmids\": [\"16709803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signal linking cell activation to tail-dependent export unknown\", \"Whether 14-3-3 binding is phosphorylation-regulated not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Systematic analysis across lymphocyte subsets showed that all resting lymphocytes store intracellular HLA-F and mobilize it to the surface upon activation — except regulatory T cells — establishing HLA-F surface display as a universal activation marker and defining its peptide-free open conformer as the predominant surface species that interacts with classical MHC-I open conformers.\",\n      \"evidence\": \"Flow cytometry and Western blot across B, T, NK, monocyte subsets; TAP/tapasin-deficient donor PBMCs; co-IP, SPR, and tetramer colocalization\",\n      \"pmids\": [\"20865824\", \"20483783\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of HLA-F–MHC-I open conformer interaction on signaling unknown\", \"Treg-specific suppression mechanism undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of KIR3DL2 and KIR2DS4 as additional receptors for HLA-F open conformers, together with a TAP-independent cross-presentation pathway mediated by HLA-F and MHC-I open conformers, expanded the functional repertoire from inhibitory signaling to both activating and antigen-presenting roles.\",\n      \"evidence\": \"SPR, biochemical pull-down, functional NK assays; cross-presentation assays with lysosomal inhibitors and MHC-I allotype blocking\",\n      \"pmids\": [\"24018270\", \"23851683\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of KIR recognition of open conformers undefined\", \"Cross-presentation pathway's in vivo relevance unestablished\", \"Whether HLA-F itself cross-presents peptide or serves as co-receptor unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A comprehensive screen identified HLA-F open conformers as high-affinity ligands for the activating receptor KIR3DS1, establishing the HLA-F/KIR3DS1 axis as a major activating pathway for NK cell degranulation and antiviral cytokine production.\",\n      \"evidence\": \"Screening 100 HLA class I proteins, SPR, primary NK cell functional assays, HIV replication inhibition, HLA-null cell line blocking experiments\",\n      \"pmids\": [\"27455421\", \"27649529\", \"29743316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of HLA-F/KIR3DS1 complex not solved\", \"Whether KIR3DS1 distinguishes among HLA-F alleles unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mass spectrometry-based peptidome characterization of HLA-F revealed presentation of unconventionally long peptides (8–24 residues) without N-terminal anchoring but with C-terminal lysine preference, and showed complete allele-specific peptide repertoire divergence, defining a non-canonical antigen presentation mode.\",\n      \"evidence\": \"Soluble HLA technology, LC-MS peptidome from reconstituted HLA-F*01:01, *01:03, *01:04 complexes\",\n      \"pmids\": [\"30941482\", \"31717259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo T cell recognition of HLA-F-presented peptides undemonstrated\", \"Structural basis of long-peptide accommodation unknown\", \"Whether peptide-loaded HLA-F engages any receptor besides KIRs untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Biochemical reconstitution showed that peptide loading onto HLA-F open conformers abolishes KIR3DS1 binding, establishing a molecular switch: peptide-free HLA-F activates NK cells via KIR3DS1 while peptide-loaded HLA-F does not, and HIV-driven enrichment of hemoglobin-derived peptides on HLA-F provides a viral immune evasion mechanism.\",\n      \"evidence\": \"Acid elution to generate open conformers, KIR3DS1 binding assay, MS proteomics of HIV-infected CD4+ T cells, peptide fraction loading and receptor recognition assay\",\n      \"pmids\": [\"33126487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How HIV promotes hemoglobin peptide loading specifically onto HLA-F is unknown\", \"Whether other viruses exploit the same evasion mechanism untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery of a progesterone-responsive distal enhancer with an ancient polymorphism creating a GATA2 binding site that loops to the HLA-F promoter revealed tissue-specific endometrial regulation, connecting HLA-F to reproductive immunology and implantation.\",\n      \"evidence\": \"GTEx replication, reporter assay, chromatin looping analysis, progesterone treatment of decidual stromal cells\",\n      \"pmids\": [\"30245028\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of enhanced HLA-F expression during implantation for NK cell tolerance unknown\", \"Whether this enhancer operates in trophoblasts themselves untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"An unexpected non-immune function was established: HLA-F promotes glioma and trophoblast cell proliferation via metabolic reprogramming through stabilization of glycolytic enzymes HK2 and PKM2 respectively, with epistatic rescue upon enzyme silencing.\",\n      \"evidence\": \"Overexpression/knockdown, glycolysis measurements, xenograft models, ChIP-seq for PKM promoter binding, siRNA epistasis\",\n      \"pmids\": [\"33867844\", \"40251569\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How an MHC-I molecule directly regulates glycolytic enzyme expression/stability is mechanistically unexplained\", \"Whether metabolic effects occur in immune cell contexts untested\", \"Independent replication needed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of HLA-F open conformer recognition by KIR3DS1, the mechanism by which HLA-F regulates glycolytic enzymes, whether peptide-loaded HLA-F engages adaptive T cell receptors in vivo, and how the switch between open conformer and peptide-loaded states is regulated during inflammation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure of HLA-F/KIR3DS1 complex\", \"No TCR recognition of HLA-F-peptide complexes demonstrated\", \"Molecular mechanism linking HLA-F to HK2/PKM2 stabilization uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [2, 9, 11, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 17, 18, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 8, 11, 23]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 3, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 9, 10, 11, 12, 18, 19]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [21, 22]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [24, 25]}\n    ],\n    \"complexes\": [\n      \"MHC class I peptide-loading complex (TAP/calreticulin)\"\n    ],\n    \"partners\": [\n      \"KIR3DS1\",\n      \"ILT2\",\n      \"ILT4\",\n      \"KIR3DL2\",\n      \"KIR2DS4\",\n      \"B2M\",\n      \"HK2\",\n      \"PKM2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}