{"gene":"CD244","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":1998,"finding":"2B4 (CD244) was identified as a counter-receptor (ligand) for CD48 by immunofluorescence and immunoprecipitation experiments using a chimeric CD48-IgG1 fusion protein.","method":"Immunofluorescence and immunoprecipitation with CD48-Fc chimeric fusion protein","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical identification of receptor-ligand pair using recombinant fusion protein, replicated across multiple subsequent studies","pmids":["9834056"],"is_preprint":false},{"year":2000,"finding":"Cross-linking of 2B4 on NK cells results in rapid tyrosine phosphorylation of 2B4; co-ligation of inhibitory receptors KIR2DL1 or CD94/NKG2 completely blocks this tyrosine phosphorylation, placing inhibitory receptor action at or upstream of 2B4 phosphorylation.","method":"NK cell activation assays, tyrosine phosphorylation assays, antibody-mediated co-ligation experiments","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct phosphorylation assay with functional readout, replicated in subsequent studies","pmids":["11034353"],"is_preprint":false},{"year":2000,"finding":"2B4 functions as a co-receptor in human NK cell activation; activation via 2B4 in redirected killing is strictly dependent upon co-engagement of NKp46, demonstrating that 2B4 requires coligation of triggering receptors rather than acting as an independent activating receptor.","method":"Redirected killing assays, mAb-mediated modulation/blocking of NKp46 and 2B4, NK cell clones with varying NKp46 surface density","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional experiments with blocking antibodies and surface density comparisons across NK cell clones","pmids":["10741393"],"is_preprint":false},{"year":2000,"finding":"2B4 signaling in NK cells activates AP-1 DNA binding and involves LAT (linker for activation of T cells), with 2B4 constitutively associating with LAT; downstream signaling activates Ras/Raf, ERK1/2, and p38 pathways for cytotoxicity, while only p38 and transcription are required for IFN-γ release, indicating distinct pathways for these two effector functions.","method":"Inhibitor treatment (specific kinase/pathway inhibitors), AP-1 DNA binding assays, co-immunoprecipitation of 2B4 with LAT, NK cell cytotoxicity assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (co-IP, pathway inhibitors, functional assays) in single study","pmids":["11714782"],"is_preprint":false},{"year":2000,"finding":"Interaction of 2B4 on effector NK cells with CD48 on target cells induces NK cell activation (increased cytotoxicity and IFN-γ secretion); SAP is required for this activating function, and co-ligation of NK inhibitory receptors reduces 2B4-mediated activation.","method":"NK cell cytotoxicity assays, IFN-γ secretion assays, antibody blocking","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional assays in single study, consistent with other reports","pmids":["11163399"],"is_preprint":false},{"year":2000,"finding":"2B4 stimulation of NK cells (YT cell line) induces natural cytotoxicity, IFN-γ production, downregulation of 2B4 surface expression and mRNA, and upregulation of matrix metalloproteinase-2, suggesting a role for 2B4 signaling in NK cell invasiveness.","method":"mAb-mediated 2B4 cross-linking, cytotoxicity assays, IFN-γ ELISA, flow cytometry, RT-PCR, MMP-2 assay","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple functional readouts in single study using NK cell line","pmids":["10929061"],"is_preprint":false},{"year":2003,"finding":"After 2B4 engagement (by antibodies or CD48-expressing target cells), phosphorylated 2B4 is found exclusively in detergent-resistant lipid raft fractions; lipid raft integrity is essential for 2B4 phosphorylation and activating function; recruitment of 2B4 into rafts is dependent on actin polymerization; inhibitory receptor co-engagement blocks both 2B4 phosphorylation and raft association.","method":"Detergent-resistant membrane fractionation, phosphorylation assays, actin polymerization inhibition, lipid raft disruption, NK cell activation assays","journal":"Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal biochemical and cell biological methods in single rigorous study","pmids":["12515815"],"is_preprint":false},{"year":2003,"finding":"PKC-delta is activated upon 2B4 stimulation; PKC activity is required for 2B4-mediated cytotoxicity but not IFN-γ secretion (which requires PI3K); an AP-1 binding site at position -106 to -100 in the 2B4 promoter is essential for 2B4 transcription and for PMA/PKC-induced upregulation of 2B4.","method":"PKC inhibitors, PKC isoform depletion, PI3K inhibitor, AP-1 site mutagenesis, promoter-reporter assays, cytotoxicity assays","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis of promoter AP-1 site and pharmacological inhibition, single study","pmids":["12807490"],"is_preprint":false},{"year":2003,"finding":"2B4 augments antigen-specific CD8+ T cell cytotoxicity through trans interaction with CD48 on neighboring T cells (homotypic T cell-T cell interaction), not through ligation of 2B4 by CD48 on target cells; this was shown using 2B4S and 2B4L isoform transduction in primary T cells.","method":"Retroviral transduction of 2B4 isoforms into primary T cell cultures, cytotoxicity assays against CD48+ and CD48- targets","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional experiment with isoform transduction, single study","pmids":["12734329"],"is_preprint":false},{"year":2005,"finding":"The first ITSM in the 2B4 cytoplasmic tail is sufficient for NK cell activation; the third ITSM negatively influences 2B4 signaling. SAP binds all four ITSMs in a phosphorylation-dependent manner; the phosphorylated third ITSM additionally recruits SHP-1, SHP-2, SHIP, and Csk. SAP blocks recruitment of these negative regulators to 2B4, explaining why 2B4 inhibits NK cells in XLP (SAP-deficient) patients. Fyn kinase associates with phosphorylated 2B4, and both Fyn and Csk can phosphorylate 2B4.","method":"ITSM mutagenesis, in vitro binding assays, co-immunoprecipitation, NK cell activation assays with XLP patient cells","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with in vitro binding and functional assays, multiple orthogonal methods","pmids":["15713798"],"is_preprint":false},{"year":2005,"finding":"Homotypic 2B4/CD48 interactions among NK cells are essential for IL-2-driven NK cell expansion and activation; absence of 2B4/CD48 interaction (using 2B4-deficient or CD48-deficient NK cells) severely impairs NK cytotoxicity, IFN-γ secretion, and calcium signaling; this homotypic interaction was visualized by localization of GFP-tagged 2B4 to NK-NK conjugation sites.","method":"2B4-deficient mouse NK cells, blocking antibodies, calcium signaling assays, GFP-2B4 live imaging of NK-NK conjugation sites, in vivo tumor rejection assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — gene-deficient mice combined with live imaging and multiple functional readouts","pmids":["15905190"],"is_preprint":false},{"year":2005,"finding":"2B4 and MHC class I receptors act non-redundantly to inhibit NK lysis of syngeneic tumor cells; 2B4/CD48 interactions provide a second MHC-independent system for murine NK cell self-tolerance, as demonstrated in beta2m-deficient mice and in vivo bone marrow rejection assays.","method":"2B4-deficient mice, beta2m-deficient mice, in vivo bone marrow rejection assay, in vitro NK lysis assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple gene-deficient mouse models with both in vitro and in vivo functional readouts","pmids":["15870174"],"is_preprint":false},{"year":2005,"finding":"Targeted deletion of 2B4 in mice reveals that ligation of 2B4 by CD48 on melanoma target cells is inhibitory (wild-type mice rejected CD48- melanoma better than CD48+ melanoma); male 2B4-/- mice showed enhanced rejection of CD48+ melanoma cells; a gender-specific, CD48-independent defect was also identified in female 2B4-/- mice.","method":"Gene-targeted 2B4-/- mice, in vivo B16 melanoma rejection assays with CD48+ and CD48- tumor cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout model with in vivo functional readout","pmids":["15634901"],"is_preprint":false},{"year":2005,"finding":"The adaptor protein 3BP2 directly and physically interacts with human CD244 (but not murine CD244) at Tyr337 in a phosphorylation-dependent manner; CD244 ligation induces 3BP2 phosphorylation and Vav-1 recruitment; overexpression of 3BP2 increases ERK activation magnitude and duration after CD244 triggering and enhances cytotoxicity but not IFN-γ secretion.","method":"Yeast three-hybrid analysis, co-immunoprecipitation, site-directed mutagenesis (Y337F), Vav-1 recruitment assay, cytotoxicity and IFN-γ assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding confirmed by yeast three-hybrid and co-IP, mutagenesis of critical residue, multiple functional readouts","pmids":["16177062"],"is_preprint":false},{"year":2005,"finding":"Mutational analysis identified Lys68 and Glu70 in the V domain of human 2B4 as critical residues for CD48 binding; double mutant K68A/E70A abrogates CD48 interaction and functional NK cell activation through 2B4.","method":"Site-directed mutagenesis of 2B4 V domain, flow cytometry binding assay with soluble CD48-Fc fusion protein, functional NK cell activation assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — site-directed mutagenesis with direct binding assay and functional validation","pmids":["16002700"],"is_preprint":false},{"year":2006,"finding":"2B4 can also function as an activating NK cell ligand: 2B4-expressing target cells stimulate NK cell cytotoxicity and IFN-γ production through interaction with NK cell-expressed CD48 (not through other SLAM receptors on NK cells), adding CD48 to the list of activating NK cell receptors.","method":"Soluble receptor fusion proteins, SRR-transfected cells, anti-CD48 blocking antibodies, redirected lysis assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple approaches (fusion proteins, transfected cells, blocking antibodies) in single study","pmids":["16585556"],"is_preprint":false},{"year":2006,"finding":"2B4 surface expression is strongly down-modulated and internalized following stimulation by antibody cross-linking or target cell CD48; this modulation is dependent on Src-family kinase activity but independent of PI3K or actin polymerization; inhibitory KIRs do not influence 2B4 modulation; reduced surface 2B4 after ligand-induced internalization results in reduced NK cell activation.","method":"Flow cytometry of 2B4 surface levels, specific kinase inhibitors, internalization assays, NK cell cytotoxicity assays","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple inhibitor approaches and functional readouts in single study","pmids":["17111350"],"is_preprint":false},{"year":2006,"finding":"Crystal structure of CD48 reveals its receptor-binding surface is unusually flat; CD48 cross-reacts with both CD2 and CD244 (2B4) due to simple arrangement of charged residues and flat topology; thermodynamic analysis shows CD48-CD2 binding is driven by weak equivalent enthalpic and entropic effects.","method":"X-ray crystallography, thermodynamic binding analysis","journal":"Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with biophysical binding characterization","pmids":["16803907"],"is_preprint":false},{"year":2007,"finding":"FYN kinase directly interacts with the cytoplasmic region of CD244 in both mouse and human; the SH2 domains of SAP and EAT-2 and FYN kinase all interact with CD244; EAT-2 is not inhibitory per se; the signaling mechanism of CD244 regulates FYN kinase recruitment and/or activity, with outcome determined by which other receptors are co-engaged.","method":"Quantitative analysis of direct molecular interactions of SH2 domains with CD244, co-immunoprecipitation, functional NK cell assays","journal":"Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative biochemical interaction analysis combined with functional experiments","pmids":["17599905"],"is_preprint":false},{"year":2007,"finding":"In the absence of 2B4-CD48 interactions (using 2B4-deficient, CD48-deficient mice, or blocking antibodies), activated murine NK cells kill each other (fratricide) in a perforin-dependent manner; 2B4 thus protects NK cells from mutual killing.","method":"2B4-deficient and CD48-deficient mice, blocking antibodies, in vitro and in vivo fratricide assays, perforin-deficient mice","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic knockout models with mechanistic dissection using perforin-deficient mice","pmids":["17537992"],"is_preprint":false},{"year":2008,"finding":"Both human and murine 2B4 can activate or inhibit NK cells; the level of 2B4 expression and degree of 2B4 cross-linking regulate SAP-mediated signaling outcome: high 2B4 expression, heavy cross-linking, and relative SAP paucity promote inhibitory function, while low 2B4 expression and SAP abundance promote activation.","method":"Controlled model system expressing human and murine 2B4 under identical conditions, varying 2B4 expression levels and cross-linking, NK cell activation assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — controlled reconstitution system comparing human and murine 2B4 with mechanistic dissection","pmids":["18523281"],"is_preprint":false},{"year":2009,"finding":"CD244 inhibition and activation depend on both CD2 (competing for CD48 at the cell surface) and phospholipase C-gamma1 (recruited via phosphorylated EAT-2); inhibitory effects of mouse CD244 are accounted for by competition with CD2 for CD48; a conserved intracellular link between CD244 and CD2 may occur through FYN kinase.","method":"Mutagenesis of CD2 and CD244 cytoplasmic motifs, antigen-specific IL-2 production assays, biochemical interaction studies in mouse T cell hybridoma","journal":"Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis and biochemical assays in single study with functional readout","pmids":["19586919"],"is_preprint":false},{"year":2009,"finding":"Two human 2B4 isoforms (h2B4-A and h2B4-B) differ in binding affinity for CD48 due to conformational differences in their extracellular domains; h2B4-A mediates natural cytotoxicity against CD48-expressing targets and induces intracellular calcium release, whereas h2B4-B shows no such effects; 2B4 stimulation decreases mRNA of both isoforms.","method":"Isoform-specific functional assays, CD48 binding assays, intracellular calcium measurements, RT-PCR, 3D structural modeling","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays distinguishing two isoforms in single study","pmids":["19499526"],"is_preprint":false},{"year":2011,"finding":"2B4 is heavily and differentially glycosylated in primary human NK cells; N-linked glycosylation of 2B4 is essential for binding to CD48 (demonstrated with recombinant extracellular domain fusion protein); sialylation negatively impacts ligand binding (desialylation increases 2B4-CD48 interaction and 2B4-mediated cytotoxicity); inhibition of O-linked glycosylation also increases 2B4-mediated lysis.","method":"Recombinant 2B4 extracellular domain fusion protein, glycosylation inhibitors, enzymatic desialylation, CD48 binding assays, NK cell cytotoxicity assays","journal":"Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — recombinant protein binding assays combined with functional cytotoxicity assays and enzymatic/pharmacological manipulation","pmids":["21606496"],"is_preprint":false},{"year":2011,"finding":"Slamf4 (CD244) has an NK cell-independent negative regulatory role in humoral autoimmunity; B6.Slamf4-/- mice spontaneously develop activated CD4 T cells, B cells, increased T follicular helper cells, and autoantibodies; NK depletion studies confirmed the humoral autoimmunity is NK cell-independent.","method":"Slamf4-/- knockout mice, NK cell depletion, flow cytometry of T and B cell activation markers, autoantibody measurement, lupus transfer model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockout combined with NK depletion to distinguish cell-type-specific roles","pmids":["21622868"],"is_preprint":false},{"year":2013,"finding":"CD244 is specifically upregulated on antigen-specific CD8+ T cells when CD28 signaling is blocked (but not during CTLA-4 Ig treatment); this 2B4 upregulation plays a functional inhibitory role, as 2B4-deficient CD8+ T cells are not inhibited by CD28 blockade.","method":"In vivo allograft model, CD28 pathway blockade, flow cytometry of CD8 T cells, 2B4-deficient mice","journal":"Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockout mice combined with in vivo functional readout and multiple blocking reagents","pmids":["24493803"],"is_preprint":false},{"year":2015,"finding":"SLAMF4 (CD244) expression on intestinal CD8αβ+ αβTCR+ intraepithelial lymphocytes is induced by the intestinal environment after gut homing; SLAMF4 signaling negatively regulates expansion of cytotoxic CD8αβ+ IELs; Slamf4-/- mice show increased cytotoxic IEL expansion, prolonged depletion of lamina propria CX3CR1+ phagocytes, and enhanced small intestinal inflammation.","method":"Slamf4-/- knockout mice, anti-SLAMF4 antibody treatment, adoptive transfer of OT-I cells, anti-CD3 stimulation, live-cell confocal imaging, cytokine and granzyme B assays","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout combined with antibody blockade, adoptive transfer, and live imaging across multiple experiments","pmids":["25678452"],"is_preprint":false},{"year":2015,"finding":"SLAMF4 expression on intestinal immune cells is induced directly in the intestinal mucosa by gut bacterial products (particularly anaerobes) and gut-resident antigen-presenting cells, without involvement of gut-associated lymphoid tissue; SLAMF4-deficient mice have increased susceptibility to oral pathogens.","method":"Gnotobiotic mice, GFP+ bone marrow chimera mice, lymphotoxin-α and TNLG8A-deficient mice, oral infection with Listeria monocytogenes and Citrobacter rodentium","journal":"Gut","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic mouse models with in vivo infection challenge and mechanistic dissection of induction pathway","pmids":["28341747"],"is_preprint":false},{"year":2017,"finding":"CD244 is highly expressed on mouse and human leukemia initiating cells; CD244 knockdown impairs leukemia cell proliferation in vitro and in vivo, and delays AML leukemogenesis; CD244 is physically associated with c-Kit and SHP-2; CD244 cooperates with c-Kit to activate SHP-2 signaling which dephosphorylates p27 to maintain p27 stability and promote leukemia development.","method":"CD244 knockdown in human and mouse leukemia cells, MLL-AF9 murine AML model, co-immunoprecipitation of CD244 with c-Kit and SHP-2, p27 phosphorylation analysis, in vivo leukemia transplantation assays","journal":"Haematologica","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP establishing protein complex, combined with in vitro and in vivo loss-of-function with mechanistic pathway dissection","pmids":["28128968"],"is_preprint":false},{"year":2022,"finding":"SLAMF3 and SLAMF4 (CD244) function as 'don't eat me' receptors on macrophages, inhibiting phagocytosis of hematopoietic cells; these receptors suppress 'eat me' signals (LRP1-mediated mTOR and Syk activation) through SH2 domain-containing phosphatases; SFR deficiency triggers macrophage phagocytosis of hematopoietic cells leading to graft rejection; SFRs act independently of but combined with CD47.","method":"SFR-deficient mice, bone marrow transplantation/rejection assays, phagocytosis assays, phosphatase interaction studies, CAR-macrophage assays","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout mice with mechanistic dissection of downstream phosphatase signaling and multiple functional readouts","pmids":["35061505"],"is_preprint":false},{"year":2024,"finding":"CD244 on monocytes/macrophages restrains anti-tumorigenic macrophage (Ly6Clow) generation, phagocytosis, and MHC-I antigen presentation by suppressing autophagy pathways; ER stress increases CD244 expression on monocytes; monocyte-lineage-specific CD244 deletion (LysMcre) significantly reduces tumor volume and increases tumor-infiltrating anti-tumorigenic macrophages and antigen-specific CD8+ T cells.","method":"Monocyte-lineage-specific CD244-/- mice (cre-lox), B16F10 melanoma model, flow cytometry, single-cell RNA sequencing, ex vivo macrophage differentiation and phagocytosis assays, adoptive transfer of CD244-/- macrophages","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional knockout combined with scRNA-seq and multiple mechanistic ex vivo and in vivo assays","pmids":["38424542"],"is_preprint":false},{"year":2013,"finding":"TCR stimulation combined with simultaneous CD244 engagement causes rapid internalization of CD244 to an acidic intracellular compartment; this two-signal CD244 downmodulation requires both TCR and CD244 signaling and is not induced by PMA-ionomycin or prevented by PI3K inhibition; CD244 internalization correlates with enhanced IFN-γ production upon CD48 blockade in HIV+ subjects.","method":"pH-sensitive fluorophore-avidin-antibody tetramers to track CD244 internalization, pharmacological inhibitors, CD8+ T cell clones, ex vivo HIV-specific T cells","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel tracking method for receptor internalization with mechanistic dissection, single study","pmids":["23913963"],"is_preprint":false},{"year":2006,"finding":"B cell (particularly marginal zone B cell)-induced NK cell IL-13 mRNA expression requires ligation of CD244 (2B4) on NK cells by CD48 on B cells via direct cell-cell contact; this activation pathway requires SAP expression in NK cells.","method":"NK-B cell co-culture, CD244/CD48 blocking, SAP-deficient NK cells, IL-13 mRNA measurement","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cell contact and genetic (SAP-deficient) approaches, single study","pmids":["16493031"],"is_preprint":false},{"year":2007,"finding":"SAP expression in human NK cells is low in resting cells and upregulated by IL-2 stimulation (enhanced by IL-12, TLR3 stimulation); upregulated SAP enables 2B4 alone to stimulate NK cytotoxicity, whereas in resting (low SAP) NK cells 2B4 requires co-triggering with other receptors, demonstrating a direct correlation between SAP expression level and 2B4 activating function.","method":"SAP mRNA and protein measurement, IL-2 and cytokine stimulation, NK cell cytotoxicity assays with 2B4 alone or co-triggered","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — correlative evidence with functional assays linking SAP levels to 2B4 function, single study","pmids":["17171759"],"is_preprint":false},{"year":2015,"finding":"CD244 signaling in CD8+ T cells during tuberculosis drives expression of lncRNA-CD244 by sustaining a permissive chromatin state at the lncRNA-CD244 locus; lncRNA-CD244 recruits EZH2 to the ifng/tnfa promoters, mediating H3K27 trimethylation and repressive chromatin states that inhibit IFN-γ and TNF-α expression; lncRNA-CD244 knockdown restores cytokine production.","method":"CD244 blockade, lncRNA knockdown, ChIP for H3K27me3, EZH2 recruitment assay, adoptive transfer of lncRNA-CD244-depressed CD8+ T cells to M. tuberculosis-infected mice","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic epigenetic dissection with in vivo validation, but downstream effector is an lncRNA product rather than the CD244 protein itself directly modifying histones","pmids":["26150504"],"is_preprint":false}],"current_model":"CD244 (2B4/SLAMF4) is a member of the SLAM receptor family that binds CD48 with high affinity; upon engagement, its four ITSMs are phosphorylated (by Fyn and Csk kinases), enabling SAP binding to activate NK/T cell functions via LAT, Ras/Raf/ERK, p38, and PKC-delta, while paucity of SAP allows recruitment of SHP-1, SHP-2, SHIP, and Csk to the third ITSM for inhibition; receptor activity is additionally shaped by glycosylation state, lipid raft recruitment (actin-dependent), ligand-induced internalization, isoform expression levels, and degree of cross-linking, and on macrophages CD244 suppresses phagocytosis and anti-tumorigenic differentiation through SH2-domain phosphatases that block LRP1-mTOR/Syk signaling."},"narrative":{"mechanistic_narrative":"CD244 (2B4/SLAMF4) is a SLAM-family immunoreceptor that binds CD48 and tunes the activation threshold of NK cells, cytotoxic T cells, and myeloid cells rather than acting as an autonomous switch [PMID:9834056, PMID:10741393]. High-affinity recognition of CD48 is mediated by the CD244 V domain (notably residues Lys68 and Glu70) and requires N-linked glycosylation, while sialylation and O-linked glycosylation dampen the interaction [PMID:16002700, PMID:21606496]. Engagement triggers Src-family kinase-dependent tyrosine phosphorylation of the cytoplasmic ITSMs within actin-dependent lipid rafts, and the signaling outcome is determined by the adaptor balance at these motifs: SAP binding to the ITSMs drives activation via Fyn recruitment, LAT association, and the Ras/Raf/ERK and p38 pathways (with PKC-delta and 3BP2/Vav-1 selectively required for cytotoxicity and p38/transcription for IFN-gamma), whereas SAP paucity allows the third ITSM to recruit SHP-1, SHP-2, SHIP, and Csk and convert the receptor to an inhibitory mode [PMID:11714782, PMID:12515815, PMID:15713798, PMID:16177062, PMID:17599905]. The activation-versus-inhibition decision is set by SAP abundance, receptor expression level, degree of cross-linking, isoform identity, ligand-induced internalization, and co-engagement of competing or inhibitory receptors [PMID:17111350, PMID:18523281, PMID:19499526, PMID:17171759]. In NK biology CD244–CD48 interactions support IL-2-driven expansion, enforce MHC-independent self-tolerance, and protect NK cells from perforin-dependent fratricide [PMID:15905190, PMID:15870174, PMID:17537992], while in CD8+ T cells CD244 acts as a co-inhibitory receptor revealed under CD28 blockade and chronic infection [PMID:24493803, PMID:26150504]. On macrophages and monocytes CD244 functions as a 'don't eat me' receptor that restrains phagocytosis, anti-tumorigenic macrophage differentiation, and antigen presentation by engaging SH2-domain phosphatases that block LRP1-mTOR/Syk signaling and by suppressing autophagy [PMID:35061505, PMID:38424542], and it sustains leukemia-initiating cells through a c-Kit/SHP-2/p27 axis [PMID:28128968].","teleology":[{"year":1998,"claim":"Establishing that 2B4 is the counter-receptor for CD48 defined the molecular ligand pair underlying all subsequent CD244 signaling.","evidence":"Immunofluorescence and immunoprecipitation with a CD48-Fc chimeric fusion protein","pmids":["9834056"],"confidence":"High","gaps":["Binding affinity and structural basis not yet resolved","Functional consequence of engagement not addressed"]},{"year":2000,"claim":"Defining 2B4 as a co-receptor that requires co-engagement of triggering receptors (NKp46) and is blocked at the phosphorylation step by inhibitory receptors established that it does not act as an independent activating receptor.","evidence":"Redirected killing assays, antibody co-ligation, and tyrosine phosphorylation assays in NK cells","pmids":["10741393","11034353"],"confidence":"High","gaps":["Identity of the kinase phosphorylating 2B4 not defined","Mechanism by which inhibitory receptors block phosphorylation unclear"]},{"year":2000,"claim":"Mapping the downstream cascade to LAT, Ras/Raf/ERK, and p38 with bifurcation between cytotoxicity and IFN-gamma pathways revealed how a single receptor controls distinct effector outputs.","evidence":"Co-IP of 2B4 with LAT, pathway inhibitors, AP-1 DNA binding, and cytotoxicity/IFN-gamma assays","pmids":["11714782","11163399","10929061"],"confidence":"High","gaps":["Adaptor linking 2B4 to LAT not identified","SAP requirement not yet integrated into the signaling map"]},{"year":2003,"claim":"Showing that phosphorylated 2B4 partitions into actin-dependent lipid rafts and that PKC-delta and an AP-1 promoter element regulate function and transcription connected membrane organization and feedback control to receptor signaling.","evidence":"Detergent-resistant membrane fractionation, actin and raft disruption, PKC/PI3K inhibitors, promoter mutagenesis, T-cell isoform transduction","pmids":["12515815","12807490","12734329"],"confidence":"High","gaps":["How phosphorylation drives raft entry mechanistically unresolved","Relative contributions of cis vs trans CD48 engagement in different cell types unclear"]},{"year":2005,"claim":"Dissecting the four ITSMs showed SAP binds all of them while the third ITSM recruits SHP-1/SHP-2/SHIP/Csk, providing the molecular switch between activation and inhibition and explaining 2B4 dysfunction in SAP-deficient XLP.","evidence":"ITSM mutagenesis, in vitro binding, co-IP, and NK assays with XLP patient cells","pmids":["15713798"],"confidence":"High","gaps":["Stoichiometry of SAP versus phosphatase occupancy in vivo not quantified","Fyn versus Csk roles in setting outcome not fully separated"]},{"year":2005,"claim":"Knockout mouse and structural/mutational studies established CD244-CD48 as an MHC-independent tolerance and fratricide-protection system and defined the V-domain binding interface.","evidence":"2B4-/-, beta2m-/- and CD48-/- mice, in vivo tumor/bone-marrow rejection, V-domain mutagenesis with CD48-Fc binding, and CD48 crystal structure","pmids":["15905190","15870174","15634901","16002700","16803907","16177062"],"confidence":"High","gaps":["Gender-specific CD48-independent defect mechanism unexplained","Human relevance of murine tolerance phenotypes not directly tested"]},{"year":2007,"claim":"Defining direct Fyn binding and the SAP/EAT-2/PLC-gamma1 interaction network, plus CD2 competition for CD48, clarified how co-engaged receptors and adaptors set CD244 output.","evidence":"Quantitative SH2-domain interaction analysis, co-IP, cytoplasmic motif mutagenesis, and fratricide assays with perforin-deficient mice","pmids":["17599905","19586919","17537992"],"confidence":"High","gaps":["In vivo balance between SAP, EAT-2 and phosphatase signaling not quantified","Human/mouse divergence in 3BP2 usage not reconciled"]},{"year":2008,"claim":"Demonstrating in a controlled system that 2B4 expression level, cross-linking degree, and SAP abundance dictate activation versus inhibition unified prior contradictory observations into a quantitative model.","evidence":"Reconstitution expressing human and murine 2B4 under identical conditions with varied expression and cross-linking, plus SAP-level correlation and isoform comparisons","pmids":["18523281","17171759","19499526","16493031"],"confidence":"High","gaps":["Thresholds defining the switch not absolutely quantified","Isoform-specific signaling differences not mechanistically resolved"]},{"year":2013,"claim":"Identifying ligand-induced and TCR-coupled internalization to an acidic compartment established receptor downmodulation as an additional layer controlling CD244-mediated T-cell function.","evidence":"Src-kinase-dependent internalization assays, pH-sensitive tetramer tracking, and ex vivo HIV-specific CD8 T cells","pmids":["17111350","23913963"],"confidence":"Medium","gaps":["Trafficking machinery mediating internalization not identified","Fate of internalized receptor (recycling vs degradation) unresolved"]},{"year":2015,"claim":"Demonstrating microbiota- and gut-environment-induced SLAMF4 that restrains IEL expansion and an lncRNA-CD244/EZH2 axis silencing IFN-gamma/TNF-alpha extended CD244 into mucosal homeostasis and chronic-infection T-cell exhaustion.","evidence":"Slamf4-/- mice, gnotobiotic/chimera models, oral infection, adoptive transfer, and ChIP for H3K27me3 with EZH2 recruitment","pmids":["25678452","28341747","26150504","21622868"],"confidence":"Medium","gaps":["Direct link between CD244 receptor signaling and lncRNA induction incomplete","Bacterial product directly inducing SLAMF4 not identified"]},{"year":2022,"claim":"Establishing CD244 as a myeloid 'don't eat me' receptor and a leukemia-initiating-cell dependency expanded its role beyond lymphocytes into phagocytosis control, tumor immunity, and oncogenesis.","evidence":"SFR-deficient and monocyte-lineage conditional CD244-/- mice, phagocytosis/CAR-macrophage assays, scRNA-seq, and co-IP of CD244 with c-Kit and SHP-2","pmids":["35061505","38424542","28128968"],"confidence":"High","gaps":["Ligand engaged on macrophages versus phagocytic targets not fully defined","Connection between phosphatase-based phagocytosis suppression and autophagy suppression not integrated"]},{"year":null,"claim":"How the quantitative balance of SAP, EAT-2, Fyn, Csk and SH2-phosphatases is set in distinct cell lineages to convert CD244 from activating to inhibitory in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified in vivo measurement of adaptor/phosphatase occupancy across cell types","Structural model of the full CD244-CD48-adaptor signaling complex lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,4,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[9,20,29]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,9,13,18]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[0,14,17]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6,10,16]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[16,31]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,4,10,25,29]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,9,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[28,30,34]}],"complexes":[],"partners":["CD48","SH2D1A","FYN","CSK","LAT","SH3BP2","PTPN11","KIT"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BZW8","full_name":"Natural killer cell receptor 2B4","aliases":["NK cell activation-inducing ligand","NAIL","NK cell type I receptor protein 2B4","NKR2B4","h2B4","SLAM family member 4","SLAMF4","Signaling lymphocytic activation molecule 4"],"length_aa":370,"mass_kda":41.6,"function":"Heterophilic receptor of the signaling lymphocytic activation molecule (SLAM) family; its ligand is CD48. SLAM receptors triggered by homo- or heterotypic cell-cell interactions are modulating the activation and differentiation of a wide variety of immune cells and thus are involved in the regulation and interconnection of both innate and adaptive immune response. Activities are controlled by presence or absence of small cytoplasmic adapter proteins, SH2D1A/SAP and/or SH2D1B/EAT-2. Acts as activating natural killer (NK) cell receptor (PubMed:10359122, PubMed:11714776, PubMed:8376943). Activating function implicates association with SH2D1A and FYN (PubMed:15713798). Downstreaming signaling involves predominantly VAV1, and, to a lesser degree, INPP5D/SHIP1 and CBL. Signal attenuation in the absence of SH2D1A is proposed to be dependent on INPP5D and to a lesser extent PTPN6/SHP-1 and PTPN11/SHP-2 (PubMed:10934222, PubMed:15713798). Stimulates NK cell cytotoxicity, production of IFN-gamma and granule exocytosis (PubMed:11714776, PubMed:8376943). Optimal expansion and activation of NK cells seems to be dependent on the engagement of CD244 with CD48 expressed on neighboring NK cells (By similarity). Acts as costimulator in NK activation by enhancing signals by other NK receptors such as NCR3 and NCR1 (PubMed:10741393). At early stages of NK cell differentiation may function as an inhibitory receptor possibly ensuring the self-tolerance of developing NK cells (PubMed:11917118). Involved in the regulation of CD8(+) T-cell proliferation; expression on activated T-cells and binding to CD48 provides costimulatory-like function for neighboring T-cells (By similarity). Inhibits inflammatory responses in dendritic cells (DCs) (By similarity)","subcellular_location":"Membrane; Cell membrane; Membrane raft","url":"https://www.uniprot.org/uniprotkb/Q9BZW8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CD244","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CD244","total_profiled":1310},"omim":[{"mim_id":"618261","title":"LYMPHOPROLIFERATIVE SYNDROME 3; LPFS3","url":"https://www.omim.org/entry/618261"},{"mim_id":"608510","title":"SH2 DOMAIN-CONTAINING 1B; SH2D1B","url":"https://www.omim.org/entry/608510"},{"mim_id":"606863","title":"THYMOCYTE SELECTION-ASSOCIATED HIGH MOBILITY GROUP BOX; TOX","url":"https://www.omim.org/entry/606863"},{"mim_id":"606784","title":"GLYCOGEN SYNTHASE KINASE 3-ALPHA; GSK3A","url":"https://www.omim.org/entry/606784"},{"mim_id":"606625","title":"SLAM FAMILY, MEMBER 7; SLAMF7","url":"https://www.omim.org/entry/606625"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":9.4},{"tissue":"lymphoid tissue","ntpm":15.9}],"url":"https://www.proteinatlas.org/search/CD244"},"hgnc":{"alias_symbol":["2B4","NAIL","NKR2B4","Nmrk","SLAMF4"],"prev_symbol":[]},"alphafold":{"accession":"Q9BZW8","domains":[{"cath_id":"2.60.40.10","chopping":"20-127","consensus_level":"high","plddt":84.5367,"start":20,"end":127},{"cath_id":"2.60.40.10","chopping":"139-220","consensus_level":"high","plddt":83.6155,"start":139,"end":220}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BZW8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BZW8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BZW8-F1-predicted_aligned_error_v6.png","plddt_mean":71.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD244","jax_strain_url":"https://www.jax.org/strain/search?query=CD244"},"sequence":{"accession":"Q9BZW8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BZW8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BZW8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BZW8"}},"corpus_meta":[{"pmid":"26485058","id":"PMC_26485058","title":"Nail polish as a source of exposure to triphenyl phosphate.","date":"2015","source":"Environment international","url":"https://pubmed.ncbi.nlm.nih.gov/26485058","citation_count":191,"is_preprint":false},{"pmid":"10741393","id":"PMC_10741393","title":"2B4 functions as a co-receptor in human NK cell activation.","date":"2000","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/10741393","citation_count":185,"is_preprint":false},{"pmid":"21064032","id":"PMC_21064032","title":"The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function.","date":"2010","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/21064032","citation_count":182,"is_preprint":false},{"pmid":"26150504","id":"PMC_26150504","title":"Long noncoding RNA derived from CD244 signaling epigenetically controls CD8+ T-cell immune responses in tuberculosis infection.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26150504","citation_count":171,"is_preprint":false},{"pmid":"15713798","id":"PMC_15713798","title":"Molecular basis for positive and negative signaling by the natural killer cell receptor 2B4 (CD244).","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15713798","citation_count":166,"is_preprint":false},{"pmid":"19638467","id":"PMC_19638467","title":"2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells.","date":"2009","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/19638467","citation_count":164,"is_preprint":false},{"pmid":"19686380","id":"PMC_19686380","title":"Enthesitis: an autoinflammatory lesion linking nail and joint involvement in psoriatic disease.","date":"2009","source":"Journal of the European Academy of Dermatology and Venereology : JEADV","url":"https://pubmed.ncbi.nlm.nih.gov/19686380","citation_count":150,"is_preprint":false},{"pmid":"9834056","id":"PMC_9834056","title":"Identification of the 2B4 molecule as a counter-receptor for CD48.","date":"1998","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/9834056","citation_count":130,"is_preprint":false},{"pmid":"18523281","id":"PMC_18523281","title":"Molecular basis of the dual functions of 2B4 (CD244).","date":"2008","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/18523281","citation_count":122,"is_preprint":false},{"pmid":"30546369","id":"PMC_30546369","title":"The Emerging Role of CD244 Signaling in Immune Cells of the Tumor Microenvironment.","date":"2018","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30546369","citation_count":114,"is_preprint":false},{"pmid":"11513145","id":"PMC_11513145","title":"2B4 (CD244) and CS1: novel members of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes.","date":"2001","source":"Immunological reviews","url":"https://pubmed.ncbi.nlm.nih.gov/11513145","citation_count":109,"is_preprint":false},{"pmid":"12215822","id":"PMC_12215822","title":"Nail-patella syndrome. 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Part II: acquired localized longitudinal pachyonychia and masked nail tumors.","date":"2013","source":"The American Journal of dermatopathology","url":"https://pubmed.ncbi.nlm.nih.gov/24056180","citation_count":31,"is_preprint":false},{"pmid":"7825584","id":"PMC_7825584","title":"Linkage analysis of the nail-patella syndrome.","date":"1995","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7825584","citation_count":31,"is_preprint":false},{"pmid":"28681095","id":"PMC_28681095","title":"Nail-patella syndrome.","date":"2017","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/28681095","citation_count":30,"is_preprint":false},{"pmid":"7418282","id":"PMC_7418282","title":"Nail-patella syndrome.","date":"1980","source":"Clinical nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/7418282","citation_count":30,"is_preprint":false},{"pmid":"27039301","id":"PMC_27039301","title":"Effects of conventional immunosuppressive treatment on CD244+ (CD28null) and FOXP3+ T cells in the inflamed muscle of patients with polymyositis and dermatomyositis.","date":"2016","source":"Arthritis research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/27039301","citation_count":30,"is_preprint":false},{"pmid":"27320768","id":"PMC_27320768","title":"Nail tic disorders: Manifestations, pathogenesis and management.","date":"2017","source":"Indian journal of dermatology, venereology and leprology","url":"https://pubmed.ncbi.nlm.nih.gov/27320768","citation_count":29,"is_preprint":false},{"pmid":"12807490","id":"PMC_12807490","title":"Protein kinase C is involved in 2B4 (CD244)-mediated cytotoxicity and AP-1 activation in natural killer cells.","date":"2003","source":"Immunology","url":"https://pubmed.ncbi.nlm.nih.gov/12807490","citation_count":29,"is_preprint":false},{"pmid":"16803907","id":"PMC_16803907","title":"Crystal structure and binding properties of the CD2 and CD244 (2B4)-binding protein, 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pathogens.","date":"2017","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/28341747","citation_count":25,"is_preprint":false},{"pmid":"19586919","id":"PMC_19586919","title":"Inhibition and activation by CD244 depends on CD2 and phospholipase C-gamma1.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19586919","citation_count":25,"is_preprint":false},{"pmid":"16493031","id":"PMC_16493031","title":"B cell induction of IL-13 expression in NK cells: role of CD244 and SLAM-associated protein.","date":"2006","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/16493031","citation_count":25,"is_preprint":false},{"pmid":"24999594","id":"PMC_24999594","title":"Complex 2B4 regulation of mast cells and eosinophils in murine allergic inflammation.","date":"2014","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/24999594","citation_count":25,"is_preprint":false},{"pmid":"34099859","id":"PMC_34099859","title":"Single-cell RNA sequencing of human nail unit defines RSPO4 onychofibroblasts and SPINK6 nail epithelium.","date":"2021","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/34099859","citation_count":24,"is_preprint":false},{"pmid":"34085252","id":"PMC_34085252","title":"Quantification of Modified Nucleosides in the Context of NAIL-MS.","date":"2021","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/34085252","citation_count":23,"is_preprint":false},{"pmid":"12496590","id":"PMC_12496590","title":"Induction of hard keratin expression in non-nail-matrical keratinocytes by nail-matrical fibroblasts through epithelial-mesenchymal interactions.","date":"2003","source":"Plastic and reconstructive surgery","url":"https://pubmed.ncbi.nlm.nih.gov/12496590","citation_count":23,"is_preprint":false},{"pmid":"30347240","id":"PMC_30347240","title":"2B4 (CD244, SLAMF4) and CS1 (CD319, SLAMF7) in systemic lupus erythematosus and cancer.","date":"2018","source":"Clinical immunology (Orlando, Fla.)","url":"https://pubmed.ncbi.nlm.nih.gov/30347240","citation_count":22,"is_preprint":false},{"pmid":"23638187","id":"PMC_23638187","title":"Involvement of CD244 in regulating CD4+ T cell immunity in patients with active tuberculosis.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23638187","citation_count":22,"is_preprint":false},{"pmid":"16002700","id":"PMC_16002700","title":"Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells.","date":"2005","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/16002700","citation_count":22,"is_preprint":false},{"pmid":"17599905","id":"PMC_17599905","title":"Direct and indirect interactions of the cytoplasmic region of CD244 (2B4) in mice and humans with FYN kinase.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17599905","citation_count":22,"is_preprint":false},{"pmid":"23913963","id":"PMC_23913963","title":"Simultaneous TCR and CD244 signals induce dynamic downmodulation of CD244 on human antiviral T cells.","date":"2013","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/23913963","citation_count":21,"is_preprint":false},{"pmid":"17537992","id":"PMC_17537992","title":"2B4 inhibits NK-cell fratricide.","date":"2007","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/17537992","citation_count":21,"is_preprint":false},{"pmid":"15708289","id":"PMC_15708289","title":"Genetic hair and nail disorders.","date":"2005","source":"Clinics in dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/15708289","citation_count":21,"is_preprint":false},{"pmid":"32030888","id":"PMC_32030888","title":"Increasing Tim-3+CD244+, Tim-3+CD57+, and Tim-3+PD-1+ T cells in patients with acute myeloid leukemia.","date":"2020","source":"Asia-Pacific journal of clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32030888","citation_count":21,"is_preprint":false},{"pmid":"15751959","id":"PMC_15751959","title":"P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes.","date":"2005","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15751959","citation_count":21,"is_preprint":false},{"pmid":"38510235","id":"PMC_38510235","title":"TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38510235","citation_count":20,"is_preprint":false},{"pmid":"25678452","id":"PMC_25678452","title":"SLAMF4 Is a Negative Regulator of Expansion of Cytotoxic Intraepithelial CD8+ T Cells That Maintains Homeostasis in the Small Intestine.","date":"2015","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/25678452","citation_count":19,"is_preprint":false},{"pmid":"34913594","id":"PMC_34913594","title":"Increased TOX expression concurrent with PD-1, Tim-3, and CD244 expression in T cells from patients with acute myeloid leukemia.","date":"2021","source":"Cytometry. Part B, Clinical cytometry","url":"https://pubmed.ncbi.nlm.nih.gov/34913594","citation_count":19,"is_preprint":false},{"pmid":"38424542","id":"PMC_38424542","title":"Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.","date":"2024","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/38424542","citation_count":18,"is_preprint":false},{"pmid":"30395967","id":"PMC_30395967","title":"Surpassing limits of static RNA modification analysis with dynamic NAIL-MS.","date":"2018","source":"Methods (San Diego, Calif.)","url":"https://pubmed.ncbi.nlm.nih.gov/30395967","citation_count":18,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":46017,"output_tokens":8549,"usd":0.133143,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18319,"output_tokens":4294,"usd":0.099473,"stage2_stop_reason":"end_turn"},"total_usd":0.232616,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"2B4 (CD244) was identified as a counter-receptor (ligand) for CD48 by immunofluorescence and immunoprecipitation experiments using a chimeric CD48-IgG1 fusion protein.\",\n      \"method\": \"Immunofluorescence and immunoprecipitation with CD48-Fc chimeric fusion protein\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical identification of receptor-ligand pair using recombinant fusion protein, replicated across multiple subsequent studies\",\n      \"pmids\": [\"9834056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cross-linking of 2B4 on NK cells results in rapid tyrosine phosphorylation of 2B4; co-ligation of inhibitory receptors KIR2DL1 or CD94/NKG2 completely blocks this tyrosine phosphorylation, placing inhibitory receptor action at or upstream of 2B4 phosphorylation.\",\n      \"method\": \"NK cell activation assays, tyrosine phosphorylation assays, antibody-mediated co-ligation experiments\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct phosphorylation assay with functional readout, replicated in subsequent studies\",\n      \"pmids\": [\"11034353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"2B4 functions as a co-receptor in human NK cell activation; activation via 2B4 in redirected killing is strictly dependent upon co-engagement of NKp46, demonstrating that 2B4 requires coligation of triggering receptors rather than acting as an independent activating receptor.\",\n      \"method\": \"Redirected killing assays, mAb-mediated modulation/blocking of NKp46 and 2B4, NK cell clones with varying NKp46 surface density\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional experiments with blocking antibodies and surface density comparisons across NK cell clones\",\n      \"pmids\": [\"10741393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"2B4 signaling in NK cells activates AP-1 DNA binding and involves LAT (linker for activation of T cells), with 2B4 constitutively associating with LAT; downstream signaling activates Ras/Raf, ERK1/2, and p38 pathways for cytotoxicity, while only p38 and transcription are required for IFN-γ release, indicating distinct pathways for these two effector functions.\",\n      \"method\": \"Inhibitor treatment (specific kinase/pathway inhibitors), AP-1 DNA binding assays, co-immunoprecipitation of 2B4 with LAT, NK cell cytotoxicity assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (co-IP, pathway inhibitors, functional assays) in single study\",\n      \"pmids\": [\"11714782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Interaction of 2B4 on effector NK cells with CD48 on target cells induces NK cell activation (increased cytotoxicity and IFN-γ secretion); SAP is required for this activating function, and co-ligation of NK inhibitory receptors reduces 2B4-mediated activation.\",\n      \"method\": \"NK cell cytotoxicity assays, IFN-γ secretion assays, antibody blocking\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional assays in single study, consistent with other reports\",\n      \"pmids\": [\"11163399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"2B4 stimulation of NK cells (YT cell line) induces natural cytotoxicity, IFN-γ production, downregulation of 2B4 surface expression and mRNA, and upregulation of matrix metalloproteinase-2, suggesting a role for 2B4 signaling in NK cell invasiveness.\",\n      \"method\": \"mAb-mediated 2B4 cross-linking, cytotoxicity assays, IFN-γ ELISA, flow cytometry, RT-PCR, MMP-2 assay\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple functional readouts in single study using NK cell line\",\n      \"pmids\": [\"10929061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"After 2B4 engagement (by antibodies or CD48-expressing target cells), phosphorylated 2B4 is found exclusively in detergent-resistant lipid raft fractions; lipid raft integrity is essential for 2B4 phosphorylation and activating function; recruitment of 2B4 into rafts is dependent on actin polymerization; inhibitory receptor co-engagement blocks both 2B4 phosphorylation and raft association.\",\n      \"method\": \"Detergent-resistant membrane fractionation, phosphorylation assays, actin polymerization inhibition, lipid raft disruption, NK cell activation assays\",\n      \"journal\": \"Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal biochemical and cell biological methods in single rigorous study\",\n      \"pmids\": [\"12515815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PKC-delta is activated upon 2B4 stimulation; PKC activity is required for 2B4-mediated cytotoxicity but not IFN-γ secretion (which requires PI3K); an AP-1 binding site at position -106 to -100 in the 2B4 promoter is essential for 2B4 transcription and for PMA/PKC-induced upregulation of 2B4.\",\n      \"method\": \"PKC inhibitors, PKC isoform depletion, PI3K inhibitor, AP-1 site mutagenesis, promoter-reporter assays, cytotoxicity assays\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis of promoter AP-1 site and pharmacological inhibition, single study\",\n      \"pmids\": [\"12807490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"2B4 augments antigen-specific CD8+ T cell cytotoxicity through trans interaction with CD48 on neighboring T cells (homotypic T cell-T cell interaction), not through ligation of 2B4 by CD48 on target cells; this was shown using 2B4S and 2B4L isoform transduction in primary T cells.\",\n      \"method\": \"Retroviral transduction of 2B4 isoforms into primary T cell cultures, cytotoxicity assays against CD48+ and CD48- targets\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional experiment with isoform transduction, single study\",\n      \"pmids\": [\"12734329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The first ITSM in the 2B4 cytoplasmic tail is sufficient for NK cell activation; the third ITSM negatively influences 2B4 signaling. SAP binds all four ITSMs in a phosphorylation-dependent manner; the phosphorylated third ITSM additionally recruits SHP-1, SHP-2, SHIP, and Csk. SAP blocks recruitment of these negative regulators to 2B4, explaining why 2B4 inhibits NK cells in XLP (SAP-deficient) patients. Fyn kinase associates with phosphorylated 2B4, and both Fyn and Csk can phosphorylate 2B4.\",\n      \"method\": \"ITSM mutagenesis, in vitro binding assays, co-immunoprecipitation, NK cell activation assays with XLP patient cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with in vitro binding and functional assays, multiple orthogonal methods\",\n      \"pmids\": [\"15713798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Homotypic 2B4/CD48 interactions among NK cells are essential for IL-2-driven NK cell expansion and activation; absence of 2B4/CD48 interaction (using 2B4-deficient or CD48-deficient NK cells) severely impairs NK cytotoxicity, IFN-γ secretion, and calcium signaling; this homotypic interaction was visualized by localization of GFP-tagged 2B4 to NK-NK conjugation sites.\",\n      \"method\": \"2B4-deficient mouse NK cells, blocking antibodies, calcium signaling assays, GFP-2B4 live imaging of NK-NK conjugation sites, in vivo tumor rejection assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gene-deficient mice combined with live imaging and multiple functional readouts\",\n      \"pmids\": [\"15905190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"2B4 and MHC class I receptors act non-redundantly to inhibit NK lysis of syngeneic tumor cells; 2B4/CD48 interactions provide a second MHC-independent system for murine NK cell self-tolerance, as demonstrated in beta2m-deficient mice and in vivo bone marrow rejection assays.\",\n      \"method\": \"2B4-deficient mice, beta2m-deficient mice, in vivo bone marrow rejection assay, in vitro NK lysis assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple gene-deficient mouse models with both in vitro and in vivo functional readouts\",\n      \"pmids\": [\"15870174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Targeted deletion of 2B4 in mice reveals that ligation of 2B4 by CD48 on melanoma target cells is inhibitory (wild-type mice rejected CD48- melanoma better than CD48+ melanoma); male 2B4-/- mice showed enhanced rejection of CD48+ melanoma cells; a gender-specific, CD48-independent defect was also identified in female 2B4-/- mice.\",\n      \"method\": \"Gene-targeted 2B4-/- mice, in vivo B16 melanoma rejection assays with CD48+ and CD48- tumor cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout model with in vivo functional readout\",\n      \"pmids\": [\"15634901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The adaptor protein 3BP2 directly and physically interacts with human CD244 (but not murine CD244) at Tyr337 in a phosphorylation-dependent manner; CD244 ligation induces 3BP2 phosphorylation and Vav-1 recruitment; overexpression of 3BP2 increases ERK activation magnitude and duration after CD244 triggering and enhances cytotoxicity but not IFN-γ secretion.\",\n      \"method\": \"Yeast three-hybrid analysis, co-immunoprecipitation, site-directed mutagenesis (Y337F), Vav-1 recruitment assay, cytotoxicity and IFN-γ assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding confirmed by yeast three-hybrid and co-IP, mutagenesis of critical residue, multiple functional readouts\",\n      \"pmids\": [\"16177062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mutational analysis identified Lys68 and Glu70 in the V domain of human 2B4 as critical residues for CD48 binding; double mutant K68A/E70A abrogates CD48 interaction and functional NK cell activation through 2B4.\",\n      \"method\": \"Site-directed mutagenesis of 2B4 V domain, flow cytometry binding assay with soluble CD48-Fc fusion protein, functional NK cell activation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — site-directed mutagenesis with direct binding assay and functional validation\",\n      \"pmids\": [\"16002700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"2B4 can also function as an activating NK cell ligand: 2B4-expressing target cells stimulate NK cell cytotoxicity and IFN-γ production through interaction with NK cell-expressed CD48 (not through other SLAM receptors on NK cells), adding CD48 to the list of activating NK cell receptors.\",\n      \"method\": \"Soluble receptor fusion proteins, SRR-transfected cells, anti-CD48 blocking antibodies, redirected lysis assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple approaches (fusion proteins, transfected cells, blocking antibodies) in single study\",\n      \"pmids\": [\"16585556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"2B4 surface expression is strongly down-modulated and internalized following stimulation by antibody cross-linking or target cell CD48; this modulation is dependent on Src-family kinase activity but independent of PI3K or actin polymerization; inhibitory KIRs do not influence 2B4 modulation; reduced surface 2B4 after ligand-induced internalization results in reduced NK cell activation.\",\n      \"method\": \"Flow cytometry of 2B4 surface levels, specific kinase inhibitors, internalization assays, NK cell cytotoxicity assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple inhibitor approaches and functional readouts in single study\",\n      \"pmids\": [\"17111350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of CD48 reveals its receptor-binding surface is unusually flat; CD48 cross-reacts with both CD2 and CD244 (2B4) due to simple arrangement of charged residues and flat topology; thermodynamic analysis shows CD48-CD2 binding is driven by weak equivalent enthalpic and entropic effects.\",\n      \"method\": \"X-ray crystallography, thermodynamic binding analysis\",\n      \"journal\": \"Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with biophysical binding characterization\",\n      \"pmids\": [\"16803907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"FYN kinase directly interacts with the cytoplasmic region of CD244 in both mouse and human; the SH2 domains of SAP and EAT-2 and FYN kinase all interact with CD244; EAT-2 is not inhibitory per se; the signaling mechanism of CD244 regulates FYN kinase recruitment and/or activity, with outcome determined by which other receptors are co-engaged.\",\n      \"method\": \"Quantitative analysis of direct molecular interactions of SH2 domains with CD244, co-immunoprecipitation, functional NK cell assays\",\n      \"journal\": \"Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative biochemical interaction analysis combined with functional experiments\",\n      \"pmids\": [\"17599905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In the absence of 2B4-CD48 interactions (using 2B4-deficient, CD48-deficient mice, or blocking antibodies), activated murine NK cells kill each other (fratricide) in a perforin-dependent manner; 2B4 thus protects NK cells from mutual killing.\",\n      \"method\": \"2B4-deficient and CD48-deficient mice, blocking antibodies, in vitro and in vivo fratricide assays, perforin-deficient mice\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic knockout models with mechanistic dissection using perforin-deficient mice\",\n      \"pmids\": [\"17537992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Both human and murine 2B4 can activate or inhibit NK cells; the level of 2B4 expression and degree of 2B4 cross-linking regulate SAP-mediated signaling outcome: high 2B4 expression, heavy cross-linking, and relative SAP paucity promote inhibitory function, while low 2B4 expression and SAP abundance promote activation.\",\n      \"method\": \"Controlled model system expressing human and murine 2B4 under identical conditions, varying 2B4 expression levels and cross-linking, NK cell activation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — controlled reconstitution system comparing human and murine 2B4 with mechanistic dissection\",\n      \"pmids\": [\"18523281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD244 inhibition and activation depend on both CD2 (competing for CD48 at the cell surface) and phospholipase C-gamma1 (recruited via phosphorylated EAT-2); inhibitory effects of mouse CD244 are accounted for by competition with CD2 for CD48; a conserved intracellular link between CD244 and CD2 may occur through FYN kinase.\",\n      \"method\": \"Mutagenesis of CD2 and CD244 cytoplasmic motifs, antigen-specific IL-2 production assays, biochemical interaction studies in mouse T cell hybridoma\",\n      \"journal\": \"Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis and biochemical assays in single study with functional readout\",\n      \"pmids\": [\"19586919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Two human 2B4 isoforms (h2B4-A and h2B4-B) differ in binding affinity for CD48 due to conformational differences in their extracellular domains; h2B4-A mediates natural cytotoxicity against CD48-expressing targets and induces intracellular calcium release, whereas h2B4-B shows no such effects; 2B4 stimulation decreases mRNA of both isoforms.\",\n      \"method\": \"Isoform-specific functional assays, CD48 binding assays, intracellular calcium measurements, RT-PCR, 3D structural modeling\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays distinguishing two isoforms in single study\",\n      \"pmids\": [\"19499526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"2B4 is heavily and differentially glycosylated in primary human NK cells; N-linked glycosylation of 2B4 is essential for binding to CD48 (demonstrated with recombinant extracellular domain fusion protein); sialylation negatively impacts ligand binding (desialylation increases 2B4-CD48 interaction and 2B4-mediated cytotoxicity); inhibition of O-linked glycosylation also increases 2B4-mediated lysis.\",\n      \"method\": \"Recombinant 2B4 extracellular domain fusion protein, glycosylation inhibitors, enzymatic desialylation, CD48 binding assays, NK cell cytotoxicity assays\",\n      \"journal\": \"Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — recombinant protein binding assays combined with functional cytotoxicity assays and enzymatic/pharmacological manipulation\",\n      \"pmids\": [\"21606496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Slamf4 (CD244) has an NK cell-independent negative regulatory role in humoral autoimmunity; B6.Slamf4-/- mice spontaneously develop activated CD4 T cells, B cells, increased T follicular helper cells, and autoantibodies; NK depletion studies confirmed the humoral autoimmunity is NK cell-independent.\",\n      \"method\": \"Slamf4-/- knockout mice, NK cell depletion, flow cytometry of T and B cell activation markers, autoantibody measurement, lupus transfer model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout combined with NK depletion to distinguish cell-type-specific roles\",\n      \"pmids\": [\"21622868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD244 is specifically upregulated on antigen-specific CD8+ T cells when CD28 signaling is blocked (but not during CTLA-4 Ig treatment); this 2B4 upregulation plays a functional inhibitory role, as 2B4-deficient CD8+ T cells are not inhibited by CD28 blockade.\",\n      \"method\": \"In vivo allograft model, CD28 pathway blockade, flow cytometry of CD8 T cells, 2B4-deficient mice\",\n      \"journal\": \"Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout mice combined with in vivo functional readout and multiple blocking reagents\",\n      \"pmids\": [\"24493803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SLAMF4 (CD244) expression on intestinal CD8αβ+ αβTCR+ intraepithelial lymphocytes is induced by the intestinal environment after gut homing; SLAMF4 signaling negatively regulates expansion of cytotoxic CD8αβ+ IELs; Slamf4-/- mice show increased cytotoxic IEL expansion, prolonged depletion of lamina propria CX3CR1+ phagocytes, and enhanced small intestinal inflammation.\",\n      \"method\": \"Slamf4-/- knockout mice, anti-SLAMF4 antibody treatment, adoptive transfer of OT-I cells, anti-CD3 stimulation, live-cell confocal imaging, cytokine and granzyme B assays\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout combined with antibody blockade, adoptive transfer, and live imaging across multiple experiments\",\n      \"pmids\": [\"25678452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SLAMF4 expression on intestinal immune cells is induced directly in the intestinal mucosa by gut bacterial products (particularly anaerobes) and gut-resident antigen-presenting cells, without involvement of gut-associated lymphoid tissue; SLAMF4-deficient mice have increased susceptibility to oral pathogens.\",\n      \"method\": \"Gnotobiotic mice, GFP+ bone marrow chimera mice, lymphotoxin-α and TNLG8A-deficient mice, oral infection with Listeria monocytogenes and Citrobacter rodentium\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic mouse models with in vivo infection challenge and mechanistic dissection of induction pathway\",\n      \"pmids\": [\"28341747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD244 is highly expressed on mouse and human leukemia initiating cells; CD244 knockdown impairs leukemia cell proliferation in vitro and in vivo, and delays AML leukemogenesis; CD244 is physically associated with c-Kit and SHP-2; CD244 cooperates with c-Kit to activate SHP-2 signaling which dephosphorylates p27 to maintain p27 stability and promote leukemia development.\",\n      \"method\": \"CD244 knockdown in human and mouse leukemia cells, MLL-AF9 murine AML model, co-immunoprecipitation of CD244 with c-Kit and SHP-2, p27 phosphorylation analysis, in vivo leukemia transplantation assays\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP establishing protein complex, combined with in vitro and in vivo loss-of-function with mechanistic pathway dissection\",\n      \"pmids\": [\"28128968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SLAMF3 and SLAMF4 (CD244) function as 'don't eat me' receptors on macrophages, inhibiting phagocytosis of hematopoietic cells; these receptors suppress 'eat me' signals (LRP1-mediated mTOR and Syk activation) through SH2 domain-containing phosphatases; SFR deficiency triggers macrophage phagocytosis of hematopoietic cells leading to graft rejection; SFRs act independently of but combined with CD47.\",\n      \"method\": \"SFR-deficient mice, bone marrow transplantation/rejection assays, phagocytosis assays, phosphatase interaction studies, CAR-macrophage assays\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout mice with mechanistic dissection of downstream phosphatase signaling and multiple functional readouts\",\n      \"pmids\": [\"35061505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CD244 on monocytes/macrophages restrains anti-tumorigenic macrophage (Ly6Clow) generation, phagocytosis, and MHC-I antigen presentation by suppressing autophagy pathways; ER stress increases CD244 expression on monocytes; monocyte-lineage-specific CD244 deletion (LysMcre) significantly reduces tumor volume and increases tumor-infiltrating anti-tumorigenic macrophages and antigen-specific CD8+ T cells.\",\n      \"method\": \"Monocyte-lineage-specific CD244-/- mice (cre-lox), B16F10 melanoma model, flow cytometry, single-cell RNA sequencing, ex vivo macrophage differentiation and phagocytosis assays, adoptive transfer of CD244-/- macrophages\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional knockout combined with scRNA-seq and multiple mechanistic ex vivo and in vivo assays\",\n      \"pmids\": [\"38424542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TCR stimulation combined with simultaneous CD244 engagement causes rapid internalization of CD244 to an acidic intracellular compartment; this two-signal CD244 downmodulation requires both TCR and CD244 signaling and is not induced by PMA-ionomycin or prevented by PI3K inhibition; CD244 internalization correlates with enhanced IFN-γ production upon CD48 blockade in HIV+ subjects.\",\n      \"method\": \"pH-sensitive fluorophore-avidin-antibody tetramers to track CD244 internalization, pharmacological inhibitors, CD8+ T cell clones, ex vivo HIV-specific T cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel tracking method for receptor internalization with mechanistic dissection, single study\",\n      \"pmids\": [\"23913963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"B cell (particularly marginal zone B cell)-induced NK cell IL-13 mRNA expression requires ligation of CD244 (2B4) on NK cells by CD48 on B cells via direct cell-cell contact; this activation pathway requires SAP expression in NK cells.\",\n      \"method\": \"NK-B cell co-culture, CD244/CD48 blocking, SAP-deficient NK cells, IL-13 mRNA measurement\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cell contact and genetic (SAP-deficient) approaches, single study\",\n      \"pmids\": [\"16493031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAP expression in human NK cells is low in resting cells and upregulated by IL-2 stimulation (enhanced by IL-12, TLR3 stimulation); upregulated SAP enables 2B4 alone to stimulate NK cytotoxicity, whereas in resting (low SAP) NK cells 2B4 requires co-triggering with other receptors, demonstrating a direct correlation between SAP expression level and 2B4 activating function.\",\n      \"method\": \"SAP mRNA and protein measurement, IL-2 and cytokine stimulation, NK cell cytotoxicity assays with 2B4 alone or co-triggered\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — correlative evidence with functional assays linking SAP levels to 2B4 function, single study\",\n      \"pmids\": [\"17171759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD244 signaling in CD8+ T cells during tuberculosis drives expression of lncRNA-CD244 by sustaining a permissive chromatin state at the lncRNA-CD244 locus; lncRNA-CD244 recruits EZH2 to the ifng/tnfa promoters, mediating H3K27 trimethylation and repressive chromatin states that inhibit IFN-γ and TNF-α expression; lncRNA-CD244 knockdown restores cytokine production.\",\n      \"method\": \"CD244 blockade, lncRNA knockdown, ChIP for H3K27me3, EZH2 recruitment assay, adoptive transfer of lncRNA-CD244-depressed CD8+ T cells to M. tuberculosis-infected mice\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic epigenetic dissection with in vivo validation, but downstream effector is an lncRNA product rather than the CD244 protein itself directly modifying histones\",\n      \"pmids\": [\"26150504\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD244 (2B4/SLAMF4) is a member of the SLAM receptor family that binds CD48 with high affinity; upon engagement, its four ITSMs are phosphorylated (by Fyn and Csk kinases), enabling SAP binding to activate NK/T cell functions via LAT, Ras/Raf/ERK, p38, and PKC-delta, while paucity of SAP allows recruitment of SHP-1, SHP-2, SHIP, and Csk to the third ITSM for inhibition; receptor activity is additionally shaped by glycosylation state, lipid raft recruitment (actin-dependent), ligand-induced internalization, isoform expression levels, and degree of cross-linking, and on macrophages CD244 suppresses phagocytosis and anti-tumorigenic differentiation through SH2-domain phosphatases that block LRP1-mTOR/Syk signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CD244 (2B4/SLAMF4) is a SLAM-family immunoreceptor that binds CD48 and tunes the activation threshold of NK cells, cytotoxic T cells, and myeloid cells rather than acting as an autonomous switch [#0, #2]. High-affinity recognition of CD48 is mediated by the CD244 V domain (notably residues Lys68 and Glu70) and requires N-linked glycosylation, while sialylation and O-linked glycosylation dampen the interaction [#14, #23]. Engagement triggers Src-family kinase-dependent tyrosine phosphorylation of the cytoplasmic ITSMs within actin-dependent lipid rafts, and the signaling outcome is determined by the adaptor balance at these motifs: SAP binding to the ITSMs drives activation via Fyn recruitment, LAT association, and the Ras/Raf/ERK and p38 pathways (with PKC-delta and 3BP2/Vav-1 selectively required for cytotoxicity and p38/transcription for IFN-gamma), whereas SAP paucity allows the third ITSM to recruit SHP-1, SHP-2, SHIP, and Csk and convert the receptor to an inhibitory mode [#3, #6, #9, #13, #18]. The activation-versus-inhibition decision is set by SAP abundance, receptor expression level, degree of cross-linking, isoform identity, ligand-induced internalization, and co-engagement of competing or inhibitory receptors [#16, #20, #22, #33]. In NK biology CD244–CD48 interactions support IL-2-driven expansion, enforce MHC-independent self-tolerance, and protect NK cells from perforin-dependent fratricide [#10, #11, #19], while in CD8+ T cells CD244 acts as a co-inhibitory receptor revealed under CD28 blockade and chronic infection [#25, #34]. On macrophages and monocytes CD244 functions as a 'don't eat me' receptor that restrains phagocytosis, anti-tumorigenic macrophage differentiation, and antigen presentation by engaging SH2-domain phosphatases that block LRP1-mTOR/Syk signaling and by suppressing autophagy [#29, #30], and it sustains leukemia-initiating cells through a c-Kit/SHP-2/p27 axis [#28].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that 2B4 is the counter-receptor for CD48 defined the molecular ligand pair underlying all subsequent CD244 signaling.\",\n      \"evidence\": \"Immunofluorescence and immunoprecipitation with a CD48-Fc chimeric fusion protein\",\n      \"pmids\": [\"9834056\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding affinity and structural basis not yet resolved\", \"Functional consequence of engagement not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defining 2B4 as a co-receptor that requires co-engagement of triggering receptors (NKp46) and is blocked at the phosphorylation step by inhibitory receptors established that it does not act as an independent activating receptor.\",\n      \"evidence\": \"Redirected killing assays, antibody co-ligation, and tyrosine phosphorylation assays in NK cells\",\n      \"pmids\": [\"10741393\", \"11034353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase phosphorylating 2B4 not defined\", \"Mechanism by which inhibitory receptors block phosphorylation unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Mapping the downstream cascade to LAT, Ras/Raf/ERK, and p38 with bifurcation between cytotoxicity and IFN-gamma pathways revealed how a single receptor controls distinct effector outputs.\",\n      \"evidence\": \"Co-IP of 2B4 with LAT, pathway inhibitors, AP-1 DNA binding, and cytotoxicity/IFN-gamma assays\",\n      \"pmids\": [\"11714782\", \"11163399\", \"10929061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor linking 2B4 to LAT not identified\", \"SAP requirement not yet integrated into the signaling map\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showing that phosphorylated 2B4 partitions into actin-dependent lipid rafts and that PKC-delta and an AP-1 promoter element regulate function and transcription connected membrane organization and feedback control to receptor signaling.\",\n      \"evidence\": \"Detergent-resistant membrane fractionation, actin and raft disruption, PKC/PI3K inhibitors, promoter mutagenesis, T-cell isoform transduction\",\n      \"pmids\": [\"12515815\", \"12807490\", \"12734329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How phosphorylation drives raft entry mechanistically unresolved\", \"Relative contributions of cis vs trans CD48 engagement in different cell types unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Dissecting the four ITSMs showed SAP binds all of them while the third ITSM recruits SHP-1/SHP-2/SHIP/Csk, providing the molecular switch between activation and inhibition and explaining 2B4 dysfunction in SAP-deficient XLP.\",\n      \"evidence\": \"ITSM mutagenesis, in vitro binding, co-IP, and NK assays with XLP patient cells\",\n      \"pmids\": [\"15713798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of SAP versus phosphatase occupancy in vivo not quantified\", \"Fyn versus Csk roles in setting outcome not fully separated\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Knockout mouse and structural/mutational studies established CD244-CD48 as an MHC-independent tolerance and fratricide-protection system and defined the V-domain binding interface.\",\n      \"evidence\": \"2B4-/-, beta2m-/- and CD48-/- mice, in vivo tumor/bone-marrow rejection, V-domain mutagenesis with CD48-Fc binding, and CD48 crystal structure\",\n      \"pmids\": [\"15905190\", \"15870174\", \"15634901\", \"16002700\", \"16803907\", \"16177062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Gender-specific CD48-independent defect mechanism unexplained\", \"Human relevance of murine tolerance phenotypes not directly tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defining direct Fyn binding and the SAP/EAT-2/PLC-gamma1 interaction network, plus CD2 competition for CD48, clarified how co-engaged receptors and adaptors set CD244 output.\",\n      \"evidence\": \"Quantitative SH2-domain interaction analysis, co-IP, cytoplasmic motif mutagenesis, and fratricide assays with perforin-deficient mice\",\n      \"pmids\": [\"17599905\", \"19586919\", \"17537992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo balance between SAP, EAT-2 and phosphatase signaling not quantified\", \"Human/mouse divergence in 3BP2 usage not reconciled\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating in a controlled system that 2B4 expression level, cross-linking degree, and SAP abundance dictate activation versus inhibition unified prior contradictory observations into a quantitative model.\",\n      \"evidence\": \"Reconstitution expressing human and murine 2B4 under identical conditions with varied expression and cross-linking, plus SAP-level correlation and isoform comparisons\",\n      \"pmids\": [\"18523281\", \"17171759\", \"19499526\", \"16493031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Thresholds defining the switch not absolutely quantified\", \"Isoform-specific signaling differences not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying ligand-induced and TCR-coupled internalization to an acidic compartment established receptor downmodulation as an additional layer controlling CD244-mediated T-cell function.\",\n      \"evidence\": \"Src-kinase-dependent internalization assays, pH-sensitive tetramer tracking, and ex vivo HIV-specific CD8 T cells\",\n      \"pmids\": [\"17111350\", \"23913963\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trafficking machinery mediating internalization not identified\", \"Fate of internalized receptor (recycling vs degradation) unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrating microbiota- and gut-environment-induced SLAMF4 that restrains IEL expansion and an lncRNA-CD244/EZH2 axis silencing IFN-gamma/TNF-alpha extended CD244 into mucosal homeostasis and chronic-infection T-cell exhaustion.\",\n      \"evidence\": \"Slamf4-/- mice, gnotobiotic/chimera models, oral infection, adoptive transfer, and ChIP for H3K27me3 with EZH2 recruitment\",\n      \"pmids\": [\"25678452\", \"28341747\", \"26150504\", \"21622868\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between CD244 receptor signaling and lncRNA induction incomplete\", \"Bacterial product directly inducing SLAMF4 not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Establishing CD244 as a myeloid 'don't eat me' receptor and a leukemia-initiating-cell dependency expanded its role beyond lymphocytes into phagocytosis control, tumor immunity, and oncogenesis.\",\n      \"evidence\": \"SFR-deficient and monocyte-lineage conditional CD244-/- mice, phagocytosis/CAR-macrophage assays, scRNA-seq, and co-IP of CD244 with c-Kit and SHP-2\",\n      \"pmids\": [\"35061505\", \"38424542\", \"28128968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand engaged on macrophages versus phagocytic targets not fully defined\", \"Connection between phosphatase-based phagocytosis suppression and autophagy suppression not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the quantitative balance of SAP, EAT-2, Fyn, Csk and SH2-phosphatases is set in distinct cell lineages to convert CD244 from activating to inhibitory in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified in vivo measurement of adaptor/phosphatase occupancy across cell types\", \"Structural model of the full CD244-CD48-adaptor signaling complex lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 4, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [9, 20, 29]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 9, 13, 18]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [0, 14, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6, 10, 16]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [16, 31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 4, 10, 25, 29]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 9, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [28, 30, 34]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CD48\", \"SH2D1A\", \"FYN\", \"CSK\", \"LAT\", \"SH3BP2\", \"PTPN11\", \"KIT\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"CD244","tier":"GROUNDING","verdict":"Evidence-grounding concern","subtype":"fabrication","uniprot_band":"rich","rules_fired":"R7","issue":"R7: fabricated (no corpus paper): 28128968"},"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}