{"gene":"CD28","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1987,"finding":"CD28 (Tp44) is a T-cell-specific homodimeric surface protein with homology to the immunoglobulin superfamily; its cDNA was cloned by transient COS cell expression, revealing it directs production of a disulfide-bonded homodimer.","method":"COS cell expression cloning, monoclonal antibody binding, molecular characterization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — original molecular cloning with functional protein production confirmed in transfected cells","pmids":["2825196"],"is_preprint":false},{"year":1985,"finding":"CD28 (9.3 antigen) is a disulfide-bonded dimer of a 44 kDa polypeptide that defines a novel T cell activation pathway independent of the T3/Ti complex, inducing IL-2 receptor expression and IL-2 secretion when crosslinked with TPA.","method":"Monoclonal antibody precipitation, co-modulation experiments, T cell activation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — original functional characterization with multiple orthogonal methods in primary T cells","pmids":["3159820"],"is_preprint":false},{"year":1991,"finding":"CD28 is the primary receptor for B7 on T cells; B7-CD28 interaction (direct binding, Kd ~200 nM) costimulates T cell proliferation and specifically increases IL-2 mRNA accumulation.","method":"Ig fusion protein binding assay, B7-transfected CHO cell costimulation, IL-2 transcript measurement","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — direct binding reconstitution with Kd measurement, functional validation with transfected cells","pmids":["1847722"],"is_preprint":false},{"year":1991,"finding":"B7 ligation of CD28 on T cells induces proliferation and high-level IL-2 secretion; this response is specific and abrogated by anti-B7 antibody.","method":"B7-transfected CHO cell costimulation assay, antibody blockade, cytokine measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — replicated functional finding with specific blocking controls","pmids":["1650475"],"is_preprint":false},{"year":1993,"finding":"B70 (B7-2/CD86) is a second ligand for CD28 and CTLA-4; monoclonal antibody to B70 inhibits CTLA4-Ig binding to B lymphoblastoid cells and blocks primary allogeneic MLR.","method":"cDNA cloning, CTLA4-Ig fusion protein binding inhibition, mixed lymphocyte reaction","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 — ligand identified by cDNA cloning, binding competition, and functional blockade","pmids":["7694153"],"is_preprint":false},{"year":1993,"finding":"CD28-knockout mice have impaired lectin-stimulated IL-2 production and reduced IL-2 receptor alpha expression; CD28 is required for T helper cell activity and immunoglobulin class switching but not for all T cell responses, indicating alternative costimulatory pathways exist.","method":"Genetic knockout mouse, T cell activation assays, immunoglobulin measurement, infection models","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with multiple defined phenotypic readouts","pmids":["7688139"],"is_preprint":false},{"year":1993,"finding":"CD28 receptor ligation induces tyrosine phosphorylation of specific substrates including phospholipase C gamma 1, and triggers both calcium-dependent and calcium-independent signals.","method":"Phosphorylation assays in activated T cells","journal":"Annual review of immunology","confidence":"Medium","confidence_rationale":"Tier 3 — biochemical identification of phosphorylated substrates, single method","pmids":["8386518"],"is_preprint":false},{"year":1994,"finding":"PI3K (phosphatidylinositol 3-kinase) binds directly to the phosphorylated YMNM (Y191MXM) motif in the CD28 cytoplasmic tail via the SH2 domains of the p85 subunit; mutation of Y191 abolishes PI3K binding and CD28-mediated IL-2 production.","method":"Site-directed mutagenesis, baculoviral p85 reconstitution, lipid kinase assay, HPLC, peptide competition","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — direct binding reconstituted in vitro with mutagenesis and multiple biochemical assays","pmids":["8146197"],"is_preprint":false},{"year":1994,"finding":"CD28 becomes tyrosine-phosphorylated after B7.1/CD80 engagement, inducing complex formation with PI3K via the p85 SH2 domain at the Y173 (YXXM) motif; CD28 mutants lacking this site cannot stimulate IL-2 production.","method":"Tyrosine phosphorylation assay, co-immunoprecipitation, CD28 mutagenesis, IL-2 production assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with direct biochemical binding and functional readout, independently replicated","pmids":["8183372"],"is_preprint":false},{"year":1994,"finding":"CD28 associates with PI3K following CD28 cross-linking, and a synthetic peptide representing the YMNM motif from the CD28 cytoplasmic tail interacts with PI3K only when the tyrosine is phosphorylated.","method":"Co-immunoprecipitation in Jurkat cells, phosphopeptide binding assay","journal":"International immunology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with phosphopeptide confirmation, single lab","pmids":["8080844"],"is_preprint":false},{"year":1995,"finding":"The CD28 pYMNM motif Y191 mutation disrupts both PI3K and Grb2 binding; M194 mutation disrupts only PI3K binding; both mutants fail to support IL-2 production upon B7-2 (CD86) engagement, directly implicating PI3K in CD28-mediated costimulation.","method":"CD28 point mutant expression, PI3K and Grb2 binding assays, IL-2 production assay with B7-2 transfected cells","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1 — selective mutagenesis dissecting binding partners with functional readout","pmids":["7584133"],"is_preprint":false},{"year":1996,"finding":"CD28 costimulation upregulates Bcl-xL expression to enhance T cell survival during activation; CD28-deficient T cells show increased apoptosis that can be blocked by ICE protease inhibitors; this survival function is Fas-independent.","method":"CD28-KO mouse T cells, Bcl-xL/Bcl-2/Fas protein expression, apoptosis assays, lpr mouse comparison, ICE protease inhibitor treatment","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with multiple orthogonal methods and specific molecular mechanism identified","pmids":["8752911"],"is_preprint":false},{"year":2000,"finding":"Cbl-b, an adaptor molecule, regulates the CD28 dependence of T cell activation; Cbl-b-deficient T cells do not require CD28 engagement for IL-2 production. Cbl-b selectively suppresses TCR-mediated Vav activation, and Cbl-b loss enhances Vav (GEF for Rac1/Rho/CDC42) activation.","method":"Cbl-b knockout mice, IL-2 production assay, genetic epistasis with CD28-KO mice, Vav activation assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with double-KO rescue and defined molecular pathway","pmids":["10646609"],"is_preprint":false},{"year":2001,"finding":"CD28 costimulation prevents anergy and promotes T cell proliferation via a PI3K-independent pathway; a Y170F point mutation in CD28 uncouples SH2-dependent signaling (PI3K/Grb2/Gads) from CD28, abolishing Bcl-xL upregulation and survival but preserving anergy prevention, proliferation, IL-2 secretion, and B cell help.","method":"Transgenic knockin mouse expressing CD28-Y170F mutant, T cell activation assays, Bcl-xL expression, anergy assessment","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — knockin mouse with point mutation separating distinct signaling outputs","pmids":["11276203"],"is_preprint":false},{"year":2001,"finding":"SIV and HIV-1 Nef proteins downmodulate CD28 by accelerating its endocytosis via the AP-2 clathrin adaptor pathway; Nef, AP-2, and CD28 form a ternary complex involving the membrane-proximal CD28 cytoplasmic domain, and Nef mutations disrupting AP-2 interaction abrogate CD28 downregulation.","method":"Nef-GFP fusion protein co-localization, co-immunoprecipitation with AP-2, endocytosis assay, Nef mutagenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — mechanistic mutagenesis combined with co-localization and co-IP evidence","pmids":["11285224"],"is_preprint":false},{"year":2001,"finding":"CD28 amplifies TCR signaling by enhancing T cell–APC contacts and specifically boosting PLCgamma1 activation and Ca2+ response downstream of TCR, without increasing Zap-70 or LAT phosphorylation; the PTK Itk controls the PLCgamma1 amplification function.","method":"Biochemical analysis of signaling intermediates (phospho-Zap70, phospho-LAT, PLCgamma1 activation, Ca2+ flux), Itk involvement assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal biochemical readouts defining mechanism","pmids":["11754815"],"is_preprint":false},{"year":2001,"finding":"TNF-alpha directly suppresses CD28 gene transcription by inhibiting activity of the CD28 minimal promoter through reduction of DNA-protein complex formation at initiator sequence motifs (sites alpha and beta), as shown by reporter gene assays and in vitro transcription assays.","method":"Reporter gene bioassay, in vitro transcription assay, EMSA (DNA-protein complex formation), nuclear extract analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple complementary in vitro methods with molecular mechanism at promoter level","pmids":["11544310"],"is_preprint":false},{"year":2002,"finding":"CD28 signaling through PI3K and Akt is required for T cells to increase glycolytic rate and glucose uptake upon activation; CD28 costimulation drives glycolytic flux in excess of immediate ATP/ADP needs, preparing cells for sustained metabolic demands.","method":"Glucose uptake assays, glycolysis measurements, PI3K/Akt inhibitor treatment, CD28-deficient T cell comparison","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — multiple metabolic assays with genetic and pharmacological perturbation of PI3K/Akt pathway","pmids":["12121659"],"is_preprint":false},{"year":2002,"finding":"Lck undergoes autophosphorylation in the immunological synapse when T cells encounter APCs; CD4 recruits Lck to the T cell–APC interface, whereas CD28 sustains Lck activation; TCR cross-linking alone cannot stimulate Lck autophosphorylation.","method":"Phosphospecific anti-Lck antiserum, APC stimulation, CD4 and CD28 perturbation, immunofluorescence","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — phosphospecific reagents with mechanistic dissection of receptor contributions","pmids":["11828322"],"is_preprint":false},{"year":2001,"finding":"CD28 co-localizes with protein kinase C theta (PKCtheta) within TCR-CD28 microclusters at the immunological synapse, and this association promotes sustained T cell signaling from a specialized subregion of the IS after mature synapse formation.","method":"Live cell imaging, microcluster analysis, immunological synapse imaging","journal":"Immunological reviews","confidence":"Medium","confidence_rationale":"Tier 3 — localization with functional implication, single lab review synthesis","pmids":["19426213"],"is_preprint":false},{"year":2004,"finding":"Soluble CD28 signals through B7-1 (CD80) and B7-2 (CD86) on dendritic cells to induce IL-6 and IFN-gamma production; IL-6 production requires CD80, CD86, and p38 MAPK and prevents IFN-gamma-driven immunosuppressive tryptophan catabolism, demonstrating reverse (inside-out) B7 signaling.","method":"Soluble CD28 treatment of DCs, cytokine measurement, p38 MAPK inhibition, in vivo adjuvant assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including pharmacological inhibition and in vivo validation","pmids":["15467723"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of soluble CD28 in complex with a mitogenic antibody Fab fragment reveals CD28 is a monovalent homodimer, redefining evolutionary relationships within the CD28/CTLA-4 superfamily and accounting for distinct ligand-binding and stoichiometric properties versus CTLA-4; cryo-EM comparisons of mitogenic vs. non-mitogenic antibody complexes constrain receptor-triggering models.","method":"X-ray crystallography, cryo-electron microscopy","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus cryo-EM, structurally validated functional differences","pmids":["15696168"],"is_preprint":false},{"year":2002,"finding":"B7-1 and B7-2 differ in affinity and structure: B7-2 binds CD28 and CTLA-4 more weakly than B7-1; relative to CTLA-4 binding, B7-2 binds CD28 2–3-fold more effectively than B7-1; B7-1 is bivalent (self-associates) while B7-2 does not; CD28 homodimers are monovalent whereas CTLA-4 homodimers are bivalent.","method":"Surface plasmon resonance, analytical ultracentrifugation, structural comparison","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1 — biophysical binding measurements with structural validation","pmids":["12196291"],"is_preprint":false},{"year":2005,"finding":"CD28 costimulation induces stable epigenetic modification of the IL-2 locus: histone acetylation, cytosine demethylation, and chromatin remodeling to an accessible state; TCR activation without CD28 fails to promote these epigenetic changes, leaving the IL-2 promoter inaccessible in anergic cells.","method":"Chromatin accessibility assay, bisulfite sequencing (cytosine methylation), ChIP (histone acetylation), comparison of activated vs. anergic T cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal epigenetic assays with clean anergy comparison","pmids":["15814687"],"is_preprint":false},{"year":2005,"finding":"CD28 engagement induces protein arginine methyltransferase activity and arginine methylation of several proteins including Vav1; methylated Vav1 localizes selectively to the nucleus; inhibiting transmethylation reduces Vav1 methylation and IL-2 production.","method":"Protein arginine methyltransferase activity assay, arginine methylation detection, subcellular fractionation, pharmacological inhibition of S-adenosyl-L-homocysteine hydrolase","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — novel PTM identified with mechanistic inhibitor and localization data","pmids":["16061726"],"is_preprint":false},{"year":2009,"finding":"p85beta (regulatory subunit of PI3K) binds to CD28 and to CBL with greater affinity than p85alpha; deletion of p85beta impairs CD28-induced c-CBL and CBL-b downregulation and PI3K pathway activation, resulting in defective T cell differentiation and failure of secondary immune response.","method":"p85beta-KO mice, co-immunoprecipitation, PI3K activation assay, CBL expression measurement, secondary immune response assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — KO mouse combined with co-IP and functional assays","pmids":["19190244"],"is_preprint":false},{"year":2002,"finding":"Genomic analysis shows CD28 agonists alone induce few gene expression changes; the principal effect of CD28 costimulation is amplification of the CD3 transcriptional response; CD28 promotes phosphorylation/inactivation of GSK3 (NFAT nuclear export kinase), enhancing nuclear NFAT accumulation and NFAT target gene expression.","method":"cDNA microarray, GSK3 phosphorylation assay, NFAT dephosphorylation assay, FK506 (calcineurin inhibitor) treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — microarray plus biochemical pathway validation with pharmacological controls","pmids":["12195013"],"is_preprint":false},{"year":2011,"finding":"B7-H2 (ICOSL) functions as a ligand for CD28 and CTLA-4 in humans (but not mice); B7-H2–CD28 interaction uses a distinct binding domain from B7-1/B7-2; B7-H2 costimulation via CD28 induces Bcl-xL, downregulates p27(kip1), and activates ERK and AKT signaling pathways.","method":"Receptor array assay, binding domain mapping, T cell costimulation assays, signaling pathway analysis (ERK, AKT phosphorylation), Bcl-xL and p27 expression","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — ligand discovery by receptor array with multiple orthogonal functional validations","pmids":["21530327"],"is_preprint":false},{"year":2013,"finding":"CD28 has an obligate cell-intrinsic function in Tregs beyond thymic development; Treg-specific Cd28 conditional knockout mice develop severe autoimmunity despite normal FOXP3+ cell numbers; CD28 loss in Tregs dampens CTLA-4, PD-1, and CCR6 expression and confers a proliferative/survival disadvantage in competitive environments.","method":"Treg-specific conditional CD28 knockout mice, flow cytometry, in vivo autoimmune models (EAE), Treg complementation experiments","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with multiple phenotypic readouts and rescue experiments","pmids":["23281398"],"is_preprint":false},{"year":2014,"finding":"CD28 and CD3 provide complementary functions in T cell force generation: force generation is associated with CD3/TCR complex engagement, while CD28 increases traction forces associated with CD3 through the PI3K signaling pathway rather than by direct cell-substrate coupling; phosphorylated Pyk2 is concentrated at sites of force generation.","method":"Traction force microscopy on elastomer pillar arrays, PI3K inhibitor treatment, phospho-Pyk2 localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — quantitative biophysical assay with pharmacological pathway dissection","pmids":["24469820"],"is_preprint":false},{"year":2016,"finding":"Bacterial superantigens bind both B7-2 and CD28 at their homodimer interfaces using the same 12-aa domain, potently enhancing B7-2/CD28 avidity and inducing T cell hyperactivation; short B7-2 dimer interface mimetic peptides prevent superantigen binding and protect mice from lethal challenge.","method":"Binding assays, B7-2 and CD28 homodimer interface mutagenesis, cytokine production assays, mouse lethality model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — mechanistic mutagenesis with in vivo validation","pmids":["27708164"],"is_preprint":false},{"year":2017,"finding":"CD28 conformation and signaling are regulated by two counteractive charged factors: acidic phospholipids sequester CD28 cytoplasmic signaling motifs within the membrane (limiting basal signaling), while TCR-induced local Ca2+ increase directly disrupts CD28-lipid interaction, releasing the cytoplasmic domain for signaling; TCR, Ca2+, and CD28 form a dual-positive-feedback circuit amplifying T cell signaling.","method":"NMR spectroscopy of CD28 transmembrane/cytoplasmic domain, lipid binding assays, Ca2+ perturbation experiments, T cell signaling assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with mechanistic functional validation","pmids":["29058713"],"is_preprint":false},{"year":2017,"finding":"CD28 transiently promotes expression of CPT1a (carnitine palmitoyltransferase 1a) before the first cell division, facilitating mitochondrial fatty acid oxidation, cristae remodeling, and enhanced spare respiratory capacity; miR-33 (targeted by TXNIP) attenuates Cpt1a in the absence of CD28; early CD28-dependent mitochondrial priming is essential for memory T cell formation.","method":"CD28-deficient T cells, Cpt1a expression measurement, miR-33 and TXNIP analysis, mitochondrial imaging, spare respiratory capacity assay, cytokine production upon restimulation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal metabolic and molecular assays in KO context with temporal resolution","pmids":["28919076"],"is_preprint":false},{"year":2016,"finding":"CTLA4-CD28 gene fusion (encoding CTLA4 extracellular domain fused to CD28 cytoplasmic domain) is frequently found in angioimmunoblastic T-cell lymphoma (58%), PTCL-NOS (23%), and extranodal NK/T-cell lymphoma (29%); ectopic expression in Jurkat/H9 cells enhances proliferation and activates AKT and ERK downstream of what would normally be an inhibitory CTLA4 signal.","method":"RNA sequencing, RT-PCR/Sanger sequencing, ectopic expression in T cell lines, AKT/ERK phosphorylation assay","journal":"Haematologica","confidence":"High","confidence_rationale":"Tier 2 — functional validation of gene fusion in multiple cell lines with signaling readout","pmids":["26819049"],"is_preprint":false},{"year":2021,"finding":"CARs containing the CD28 transmembrane domain (TMD) heterodimerize with endogenous CD28 in human T cells via polar amino acids in the CD28-TMD; this heterodimerization reduces CD28 cell-surface expression and does not respond to CD80/CD86 stimulation, revealing a fundamental functional difference from CD8-TMD CARs.","method":"Co-immunoprecipitation, CAR-dependent proliferation assay upon anti-CD28 stimulation, CD28-TMD mutagenesis, flow cytometry","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 — co-IP with mutagenesis and functional validation","pmids":["33833759"],"is_preprint":false},{"year":2023,"finding":"CD8+ T cells display B7 ligands (CD80/CD86) that interact with CD28 in cis at invaginated synaptic membranes; this cis-B7:CD28 interaction is driven by PI3K and sorting nexin-9 (SNX9), triggers CD28 signaling through PKCtheta, and promotes CD8+ T cell survival, migration, and cytokine production; loss of cis-B7:CD28 interactions in mouse tumor models decreases intratumoral T cells and accelerates tumor growth.","method":"Live cell imaging, co-IP, PI3K and SNX9 perturbation, PKCtheta signaling assay, mouse tumor models with T cell-specific B7 knockout","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — mechanistic dissection with multiple orthogonal methods and in vivo validation","pmids":["37160118"],"is_preprint":false},{"year":2020,"finding":"CD28 transduces pro-survival signaling in long-lived plasma cells (LLPCs) specifically through differential SLP76 expression; CD28 signaling in LLPCs increases glucose uptake, mitochondrial mass/respiration, and ROS production; CD28-mediated NF-κB activation and survival are ROS-dependent; IRF4 (NF-κB target) upregulated by CD28 mediates this metabolic program.","method":"LLPC vs. SLPC comparison, SLP76 expression analysis, glucose uptake, mitochondrial assays, ROS measurement, NF-κB activation assay, IRF4 expression","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal metabolic and signaling assays with mechanistic dissection","pmids":["32579940"],"is_preprint":false},{"year":2024,"finding":"CD28 costimulatory domain incorporated into CAR-NK cells recruits LCK and ZAP70 kinases to the CD3ζ signaling complex, initiating a signaling cascade that enhances CAR-NK cell antitumor persistence and cytotoxicity.","method":"CAR-NK cell engineering, kinase recruitment assay, in vitro cytotoxicity assay, multiple xenograft mouse models","journal":"Cancer discovery","confidence":"High","confidence_rationale":"Tier 2 — mechanistic kinase recruitment demonstrated with in vitro and in vivo validation","pmids":["38900051"],"is_preprint":false},{"year":1997,"finding":"Itk negatively regulates CD28-mediated T cell proliferation: Itk-deficient T cells show augmented proliferative response to CD28 costimulation (calcineurin-independent) without increased IL-2, establishing that Itk plays distinct inhibitory roles in CD28 versus CD3 signaling pathways.","method":"Itk-knockout mice, T cell proliferation assay, IL-2 measurement, calcineurin pathway inhibition","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with defined pathway dissection","pmids":["9221751"],"is_preprint":false},{"year":2000,"finding":"CD28 can activate PI3K and cooperate with adapters Vav and SLP-76 to induce IL-2 and IL-4 transcription independently of TCR ligation, providing a molecular basis for in trans costimulation.","method":"CD28 stimulation without TCR ligation, IL-2/IL-4 promoter assays, Vav and SLP-76 co-signaling analysis","journal":"Immunological reviews","confidence":"Medium","confidence_rationale":"Tier 3 — pathway characterization, single method per component","pmids":["12670393"],"is_preprint":false},{"year":2000,"finding":"Unexpectedly, the Y189F CD28 YMNM mutant that disrupts PI3K binding shows reduced but significant IL-2 promoter activity, while N191A (retains PI3K binding) completely abrogates activity; PI3K inhibitor augments IL-2 promoter activity while constitutively active PI3K reduces it, suggesting PI3K acts as a negative mediator of CD28-mediated IL-2 transcription.","method":"CD28 YMNM point/deletion mutant expression in Jurkat, IL-2 promoter reporter assay, PI3K pharmacological inhibitor, constitutively active PI3K overexpression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple mutants and pharmacological tools, but finding contradicts other studies (context-dependent)","pmids":["11113113"],"is_preprint":false},{"year":2013,"finding":"CD28 co-stimulatory pathway, specifically through the Tec-family kinase ITK, regulates trafficking of autoreactive T cells to tissues; ablation of ITK in Ctla4−/− mice blocks tissue infiltration without preventing lymphoid organ T cell activation; ITK inhibitors phenocopy ITK null and prevent islet infiltration in diabetes models.","method":"Double-KO mice (Itk−/−;Ctla4−/−), T cell trafficking analysis, ITK pharmacological inhibition, pancreatic islet infiltration model","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with pharmacological validation and in vivo disease model","pmids":["24270545"],"is_preprint":false},{"year":2016,"finding":"CD28 costimulation drives distinct metabolic programs in CAR T cells: CD28 signaling domains favor effector memory differentiation with a glycolytic gene signature, while 4-1BB domains promote central memory with enhanced fatty acid oxidation and mitochondrial biogenesis.","method":"Metabolic flux analysis, gene expression profiling, mitochondrial assays in CAR T cells with different costimulatory domains","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — multiple metabolic assays with direct CD28 vs. 4-1BB domain comparison","pmids":["26885860"],"is_preprint":false}],"current_model":"CD28 is a disulfide-bonded homodimeric T cell costimulatory receptor that, upon engagement by B7-1 (CD80), B7-2 (CD86), or B7-H2 (in humans), becomes tyrosine-phosphorylated at its cytoplasmic YMNM motif, recruiting PI3K (p85/p110) and adaptor proteins (Grb2, Vav, SLP-76) to activate PI3K–Akt signaling, boost PLCγ1 and Ca2+/NFAT responses, inactivate GSK3, induce arginine methylation of Vav1, upregulate Bcl-xL for survival, drive glycolysis and mitochondrial fatty acid oxidation, and promote epigenetic remodeling of the IL-2 locus—with CD28 transmembrane signaling further regulated by membrane lipid sequestration relieved by TCR-induced Ca2+, and cis-B7:CD28 interactions at invaginated synaptic membranes extending costimulation in peripheral tissues."},"narrative":{"teleology":[{"year":1985,"claim":"Identification of CD28 (9.3 antigen) as a disulfide-bonded homodimeric T cell surface protein defining a costimulatory activation pathway independent of the TCR/CD3 complex resolved the question of whether T cell activation requires signals beyond the antigen receptor.","evidence":"Monoclonal antibody precipitation, co-modulation experiments, and T cell activation assays in primary human T cells","pmids":["3159820"],"confidence":"High","gaps":["Ligand identity unknown","Signaling mechanism undefined","In vivo relevance not tested"]},{"year":1987,"claim":"Molecular cloning of CD28 cDNA established it as an immunoglobulin superfamily member and confirmed its homodimeric structure, enabling all subsequent structure-function studies.","evidence":"COS cell expression cloning with monoclonal antibody binding confirmation","pmids":["2825196"],"confidence":"High","gaps":["Ligand still unidentified","Cytoplasmic signaling motifs not yet mapped"]},{"year":1991,"claim":"Identification of B7 (CD80) as the cognate CD28 ligand with measured binding affinity (~200 nM) established the receptor-ligand pair governing T cell costimulation and IL-2 production.","evidence":"Ig fusion protein binding assay, B7-transfected CHO cell costimulation, IL-2 transcript quantification","pmids":["1847722","1650475"],"confidence":"High","gaps":["Possibility of additional ligands not excluded","Intracellular signaling pathway downstream of engagement unknown"]},{"year":1993,"claim":"Discovery of B7-2 (CD86) as a second CD28/CTLA-4 ligand and generation of CD28-knockout mice collectively demonstrated that CD28 is essential for robust IL-2 production and Ig class switching but that alternative costimulatory pathways exist.","evidence":"B7-2 cDNA cloning with CTLA4-Ig binding competition; CD28-KO mice with T cell activation, Ig, and infection phenotyping","pmids":["7694153","7688139"],"confidence":"High","gaps":["Downstream signaling intermediates not molecularly defined","Mechanism of alternative costimulation unknown"]},{"year":1994,"claim":"Mapping PI3K recruitment to the phosphorylated YMNM motif in the CD28 cytoplasmic tail via p85 SH2 domains—with Y191 mutation abolishing both PI3K binding and IL-2 production—established the central signaling axis of CD28 costimulation.","evidence":"Site-directed mutagenesis, baculoviral p85 reconstitution, lipid kinase assay, co-immunoprecipitation in multiple labs","pmids":["8146197","8183372","8080844"],"confidence":"High","gaps":["PI3K-independent CD28 functions not yet identified","Grb2 contribution not yet dissected from PI3K"]},{"year":1995,"claim":"Selective mutagenesis of the YMNM motif separated PI3K and Grb2 binding requirements, showing both are needed for full IL-2 induction and establishing that CD28 nucleates a multi-adaptor signaling complex.","evidence":"CD28 point mutants (Y191, M194) with selective PI3K/Grb2 disruption and IL-2 assay using B7-2-transfected cells","pmids":["7584133"],"confidence":"High","gaps":["Downstream targets of Grb2 in CD28 pathway not defined","Role of additional adaptors (Vav, SLP-76) not yet tested"]},{"year":1996,"claim":"Demonstration that CD28 costimulation upregulates Bcl-xL to prevent T cell apoptosis identified the survival arm of CD28 signaling as mechanistically distinct from its proliferative arm.","evidence":"CD28-KO T cells with Bcl-xL/Bcl-2 expression analysis, apoptosis assays, ICE protease inhibition","pmids":["8752911"],"confidence":"High","gaps":["Whether Bcl-xL induction requires PI3K or uses an independent pathway not resolved","Relationship to anergy prevention unclear"]},{"year":2000,"claim":"Genetic epistasis showed Cbl-b gates CD28 dependence by suppressing Vav activation; Cbl-b loss bypasses the CD28 requirement, placing Vav as a critical downstream node in CD28-dependent T cell activation.","evidence":"Cbl-b KO and Cbl-b/CD28 double-KO mice with Vav activation and IL-2 assays","pmids":["10646609"],"confidence":"High","gaps":["Mechanism by which CD28 relieves Cbl-b suppression of Vav not defined","Quantitative contribution of Vav vs. PI3K not separated"]},{"year":2001,"claim":"A Y170F CD28 knockin mouse uncoupled PI3K/Grb2-dependent signals (Bcl-xL, survival) from PI3K-independent functions (anergy prevention, proliferation, IL-2), demonstrating that CD28 engages parallel signaling branches with separable biological outcomes.","evidence":"Transgenic knockin mouse expressing CD28-Y170F, T cell activation and anergy assays","pmids":["11276203"],"confidence":"High","gaps":["Identity of the PI3K-independent signaling pathway not molecularly resolved","In vivo infection/autoimmune consequences not fully explored"]},{"year":2001,"claim":"CD28 was shown to amplify TCR signaling specifically at the PLCγ1/Ca²⁺ node (via Itk) without increasing proximal Zap-70 or LAT phosphorylation, defining the point of signal integration between TCR and CD28.","evidence":"Biochemical analysis of phospho-Zap70, phospho-LAT, PLCγ1 activation, and Ca²⁺ flux with Itk perturbation","pmids":["11754815"],"confidence":"High","gaps":["How CD28 activates Itk at the molecular level not shown","Whether this applies to all T cell subsets unknown"]},{"year":2002,"claim":"Genomic and biochemical studies revealed that CD28 primarily amplifies CD3-initiated transcription rather than activating unique genes, with GSK3 inactivation promoting NFAT nuclear accumulation as a key mechanism, while CD28-PI3K-Akt drives glycolysis and glucose uptake to meet metabolic demands of activation.","evidence":"cDNA microarray, GSK3 phosphorylation assay, glucose uptake and glycolysis measurements with PI3K/Akt perturbation","pmids":["12195013","12121659"],"confidence":"High","gaps":["Whether GSK3 inactivation is PI3K-dependent or PI3K-independent in this context not fully resolved","Metabolic requirements in different T cell subsets not compared"]},{"year":2002,"claim":"Biophysical characterization established that CD28 homodimers are monovalent for ligand binding whereas CTLA-4 is bivalent, with B7-2 preferentially binding CD28 over CTLA-4 relative to B7-1, explaining differential costimulatory versus inhibitory receptor engagement.","evidence":"Surface plasmon resonance and analytical ultracentrifugation","pmids":["12196291"],"confidence":"High","gaps":["How monovalent binding translates to signaling threshold not modeled","In vivo kinetic competition between CD28 and CTLA-4 not measured"]},{"year":2005,"claim":"Crystal structure of CD28 confirmed the monovalent homodimer architecture and, combined with cryo-EM of mitogenic versus non-mitogenic antibody complexes, constrained models of receptor triggering geometry.","evidence":"X-ray crystallography and cryo-electron microscopy","pmids":["15696168"],"confidence":"High","gaps":["No structure of CD28 bound to B7 ligands available","Transmembrane and cytoplasmic domain conformational changes not captured"]},{"year":2005,"claim":"CD28 costimulation was shown to induce stable epigenetic remodeling of the IL-2 locus (histone acetylation, CpG demethylation, chromatin opening), establishing that CD28 programs the epigenetic state distinguishing activated from anergic T cells.","evidence":"Chromatin accessibility, bisulfite sequencing, ChIP for histone marks in activated versus anergic T cells","pmids":["15814687"],"confidence":"High","gaps":["Specific chromatin remodelers recruited by CD28 signaling not identified","Whether epigenetic effects are direct or secondary to transcription factor binding unclear"]},{"year":2005,"claim":"Discovery that CD28 engagement induces arginine methylation of Vav1, redirecting it to the nucleus, identified a novel post-translational modification linking CD28 to nuclear signaling.","evidence":"PRMT activity assay, arginine methylation detection, subcellular fractionation, transmethylation inhibitor","pmids":["16061726"],"confidence":"High","gaps":["Which PRMT is responsible not identified","Nuclear function of methylated Vav1 not characterized"]},{"year":2011,"claim":"Identification of B7-H2 (ICOSL) as a third human CD28 ligand using a distinct binding site expanded the ligand repertoire and showed B7-H2:CD28 activates Akt/ERK and upregulates Bcl-xL.","evidence":"Receptor array, binding domain mapping, costimulation assays, signaling pathway analysis","pmids":["21530327"],"confidence":"High","gaps":["Not conserved in mice, limiting genetic model validation","Physiological contexts where B7-H2:CD28 dominates over B7-1/B7-2 not defined"]},{"year":2013,"claim":"Treg-specific conditional CD28 deletion revealed a cell-intrinsic post-developmental role for CD28 in maintaining Treg homeostasis and suppressive function, with loss causing severe autoimmunity despite normal Treg numbers.","evidence":"Treg-specific CD28 conditional KO mice, flow cytometry, EAE model, complementation experiments","pmids":["23281398"],"confidence":"High","gaps":["Downstream molecular program by which CD28 maintains Treg suppression not fully mapped","Whether CTLA-4 downregulation is cause or consequence of Treg failure unclear"]},{"year":2017,"claim":"NMR studies revealed that acidic phospholipids sequester the CD28 cytoplasmic domain at the membrane, with TCR-induced Ca²⁺ disrupting this interaction to liberate signaling motifs—establishing a membrane-level gating mechanism for CD28 activation.","evidence":"NMR spectroscopy of CD28 TM/cytoplasmic domain, lipid binding assays, Ca²⁺ perturbation, T cell signaling readouts","pmids":["29058713"],"confidence":"High","gaps":["Whether lipid composition variations across T cell subsets modulate this gating is unknown","In vivo confirmation of the dual feedback circuit not yet achieved"]},{"year":2017,"claim":"CD28 was shown to transiently induce CPT1a before first division, priming mitochondrial fatty acid oxidation and spare respiratory capacity essential for memory T cell formation, with miR-33 attenuating this program in the absence of CD28.","evidence":"CD28-deficient T cells, CPT1a expression kinetics, miR-33/TXNIP analysis, mitochondrial imaging, spare respiratory capacity measurement","pmids":["28919076"],"confidence":"High","gaps":["Signaling branch (PI3K vs. PI3K-independent) driving CPT1a induction not identified","Whether this metabolic priming applies to CD4⁺ T cells equally is unknown"]},{"year":2020,"claim":"CD28 was found to transduce pro-survival signals in long-lived plasma cells through SLP-76-dependent NF-κB activation driving IRF4 and a ROS-dependent metabolic program, extending CD28 function beyond T cells.","evidence":"LLPC vs. SLPC comparison, SLP76 expression, glucose uptake, mitochondrial/ROS assays, NF-κB and IRF4 measurement","pmids":["32579940"],"confidence":"High","gaps":["CD28 ligand source in the bone marrow niche not defined","Whether plasma cell CD28 signaling uses the same YMNM-PI3K axis as T cells not tested"]},{"year":2023,"claim":"Discovery of cis-B7:CD28 interactions on CD8⁺ T cells at invaginated synaptic membranes—driven by PI3K and SNX9 and signaling through PKCθ—revealed a cell-autonomous costimulatory mechanism that sustains intratumoral T cell survival.","evidence":"Live cell imaging, co-IP, PI3K/SNX9 perturbation, PKCθ assay, T cell-specific B7 KO in mouse tumor models","pmids":["37160118"],"confidence":"High","gaps":["Structural basis of cis vs. trans B7:CD28 interaction not resolved","Whether cis-signaling occurs in CD4⁺ T cells or Tregs unknown"]},{"year":null,"claim":"Key unresolved questions include the structural basis of the CD28–B7 ligand complex, the molecular identity of the PI3K-independent CD28 signaling branch that prevents anergy, the chromatin remodelers directly recruited by CD28 signaling, and how cis- versus trans-B7 engagement is regulated across immune cell types and tissue microenvironments.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length CD28:B7 co-crystal structure exists","PI3K-independent anergy-prevention pathway molecularly uncharacterized","Tissue-specific regulation of cis-B7:CD28 not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[1,2,3,5,7,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7,10,39]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,21,31,34,35]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,3,5,7,8,10,11,12,13,15,26,28,35]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,8,10,17,26,27,31,36]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[17,32,42]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[11,36]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[23]}],"complexes":[],"partners":["CD80","CD86","PIK3R1","GRB2","VAV1","SLP76","PRKCQ","ITK"],"other_free_text":[]},"mechanistic_narrative":"CD28 is the principal costimulatory receptor on T cells, integrating signals from B7 family ligands to amplify TCR-driven activation, promote survival, and shape metabolic and epigenetic programs that determine effector and memory T cell fate. Identified as a disulfide-bonded homodimeric immunoglobulin superfamily member, CD28 binds B7-1 (CD80), B7-2 (CD86), and—in humans—B7-H2 (ICOSL); upon ligand engagement and tyrosine phosphorylation of its cytoplasmic YMNM motif, it recruits PI3K (p85/p110), Grb2, and adaptors Vav1 and SLP-76 to activate PI3K–Akt signaling, boost PLCγ1/Ca²⁺/NFAT responses, inactivate GSK3, upregulate Bcl-xL for survival, drive glycolysis and early mitochondrial fatty acid oxidation essential for memory formation, and promote epigenetic remodeling of the IL-2 locus [PMID:8146197, PMID:7584133, PMID:12121659, PMID:28919076, PMID:15814687, PMID:8752911]. Transmembrane-level regulation involves acidic phospholipid sequestration of the cytoplasmic domain, relieved by TCR-induced Ca²⁺, creating a dual positive-feedback circuit, while cis-B7:CD28 interactions at invaginated synaptic membranes on CD8⁺ T cells sustain costimulation via PI3K/SNX9/PKCθ to promote intratumoral T cell survival [PMID:29058713, PMID:37160118]. Beyond conventional T cells, CD28 has a cell-intrinsic function in regulatory T cell homeostasis—Treg-specific CD28 deletion causes severe autoimmunity—and transduces pro-survival metabolic signaling in long-lived plasma cells through NF-κB/IRF4-dependent pathways [PMID:23281398, PMID:32579940]."},"prefetch_data":{"uniprot":{"accession":"P10747","full_name":"T-cell-specific surface glycoprotein CD28","aliases":["TP44"],"length_aa":220,"mass_kda":25.1,"function":"Receptor that plays a role in T-cell activation, proliferation, survival and the maintenance of immune homeostasis (PubMed:1650475, PubMed:7568038). Functions not only as an amplifier of TCR signals but delivers unique signals that control intracellular biochemical events that alter the gene expression program of T-cells (PubMed:24665965). Stimulation upon engagement of its cognate ligands CD80 or CD86 increases proliferation and expression of various cytokines in particular IL2 production in both CD4(+) and CD8(+) T-cell subsets (PubMed:12196291, PubMed:1650475, PubMed:35397202). Mechanistically, ligation induces recruitment of protein kinase C-theta/PRKCQ and GRB2 leading to NF-kappa-B activation via both PI3K/Akt-dependent and -independent pathways (PubMed:21964608, PubMed:24665965, PubMed:7568038). In conjunction with TCR/CD3 ligation and CD40L costimulation, enhances the production of IL4 and IL10 in T-cells (PubMed:8617933) Enhances CD40L-mediated activation of NF-kappa-B and kinases MAPK8 and PAK2 in T-cells (PubMed:15067037)","subcellular_location":"Cell surface","url":"https://www.uniprot.org/uniprotkb/P10747/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CD28","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CD28","total_profiled":1310},"omim":[{"mim_id":"621017","title":"ZINC FINGER PROTEIN 334; ZNF334","url":"https://www.omim.org/entry/621017"},{"mim_id":"620901","title":"IMMUNODEFICIENCY 123 WITH HPV-RELATED VERRUCOSIS; IMD123","url":"https://www.omim.org/entry/620901"},{"mim_id":"620678","title":"RAS AND RAB INTERACTOR-LIKE PROTEIN; RINL","url":"https://www.omim.org/entry/620678"},{"mim_id":"620430","title":"AUTOIMMUNE DISEASE, MULTISYSTEM, INFANTILE-ONSET, 3; ADMIO3","url":"https://www.omim.org/entry/620430"},{"mim_id":"619164","title":"IMMUNODEFICIENCY 76; IMD76","url":"https://www.omim.org/entry/619164"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":28.7},{"tissue":"placenta","ntpm":13.9}],"url":"https://www.proteinatlas.org/search/CD28"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P10747","domains":[{"cath_id":"2.60.40.10","chopping":"22-134","consensus_level":"high","plddt":93.3325,"start":22,"end":134},{"cath_id":"1.20.5","chopping":"151-181","consensus_level":"medium","plddt":89.8632,"start":151,"end":181}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P10747","model_url":"https://alphafold.ebi.ac.uk/files/AF-P10747-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P10747-F1-predicted_aligned_error_v6.png","plddt_mean":81.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD28","jax_strain_url":"https://www.jax.org/strain/search?query=CD28"},"sequence":{"accession":"P10747","fasta_url":"https://rest.uniprot.org/uniprotkb/P10747.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P10747/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P10747"}},"corpus_meta":[{"pmid":"8717514","id":"PMC_8717514","title":"CD28/B7 system of T cell costimulation.","date":"1996","source":"Annual review of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/8717514","citation_count":2082,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11910893","id":"PMC_11910893","title":"The B7-CD28 superfamily.","date":"2002","source":"Nature reviews. 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A new activation pathway used by a major T cell population via a disulfide-bonded dimer of a 44 kilodalton polypeptide (9.3 antigen).","date":"1985","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/3159820","citation_count":362,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9784967","id":"PMC_9784967","title":"CD28/B7 costimulation: a review.","date":"1998","source":"Critical reviews in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/9784967","citation_count":351,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8183372","id":"PMC_8183372","title":"Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling.","date":"1994","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8183372","citation_count":345,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7516408","id":"PMC_7516408","title":"Interleukin 12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells.","date":"1994","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/7516408","citation_count":343,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8484124","id":"PMC_8484124","title":"Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation.","date":"1993","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8484124","citation_count":297,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19898481","id":"PMC_19898481","title":"Genetic variants at CD28, PRDM1 and CD2/CD58 are associated with rheumatoid arthritis risk.","date":"2009","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19898481","citation_count":284,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11279501","id":"PMC_11279501","title":"Structural basis for co-stimulation by the human CTLA-4/B7-2 complex.","date":"2001","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11279501","citation_count":269,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8146197","id":"PMC_8146197","title":"T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.","date":"1994","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8146197","citation_count":265,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9032261","id":"PMC_9032261","title":"Lck regulates Vav activation of members of the Rho family of GTPases.","date":"1997","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9032261","citation_count":264,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11714771","id":"PMC_11714771","title":"Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule.","date":"2001","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/11714771","citation_count":251,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20638642","id":"PMC_20638642","title":"Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses.","date":"2010","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/20638642","citation_count":250,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12195013","id":"PMC_12195013","title":"Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12195013","citation_count":243,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":39277,"output_tokens":7689,"usd":0.116583},"stage2":{"model":"claude-opus-4-6","input_tokens":11469,"output_tokens":4455,"usd":0.25308},"total_usd":0.802173,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":52240,"output_tokens":9312,"usd":0.1482},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":13503,"output_tokens":4881,"usd":0.28431}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1993,\n      \"finding\": \"CD28 is the receptor for B7/BB-1 on activated B cells, dendritic cells, and macrophages; CD28 ligation induces tyrosine phosphorylation of specific substrates including phospholipase C gamma 1, and triggers both calcium-dependent and calcium-independent signals\",\n      \"method\": \"Biochemical signaling assays, receptor-ligand binding studies\",\n      \"journal\": \"Annual review of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated across multiple labs, foundational receptor-ligand identification with downstream signaling characterization\",\n      \"pmids\": [\"8386518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"B70 (B7-2/CD86) is a second ligand for CD28 and CTLA-4, expressed on resting monocytes and dendritic cells and activated lymphocytes; anti-B70 antibody substantially inhibited CTLA4-Ig binding to B-lymphoblastoid cell lines and inhibited primary allogeneic mixed lymphocyte responses\",\n      \"method\": \"Monoclonal antibody generation, cDNA cloning, CTLA4-Ig binding inhibition assay, mixed lymphocyte reaction\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding assays with functional validation, widely replicated\",\n      \"pmids\": [\"7694153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"CD28-deficient mice show impaired T cell responses to lectins with abolished IL-2 production and reduced IL-2 receptor alpha expression; CD28 is required for T helper cell activity and immunoglobulin class switching but not all T cell responses\",\n      \"method\": \"CD28 knockout mouse generation, T cell stimulation assays, cytokine measurement, immunoglobulin isotype analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotypes, highly cited foundational paper\",\n      \"pmids\": [\"7688139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CD28 associates with phosphatidylinositol 3-kinase (PI3K) via its cytoplasmic YMNM motif upon tyrosine phosphorylation following CD28 cross-linking\",\n      \"method\": \"Co-immunoprecipitation, synthetic phosphopeptide binding assay in Jurkat cells\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical association demonstrated, replicated in multiple subsequent studies\",\n      \"pmids\": [\"8080844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"CD28 pYMNM motif Y191 mutation disrupts both PI3K and GRB-2 binding while M194 mutation disrupts only PI3K binding; both mutants abolish IL-2 production upon CD86-CD28 engagement, directly implicating PI3K in CD28-mediated costimulation leading to IL-2 secretion\",\n      \"method\": \"Site-directed mutagenesis of CD28 cytoplasmic domain, IL-2 production assay with CHO-CD86 cells\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro mutagenesis with functional readout, mechanistic dissection of PI3K binding motif\",\n      \"pmids\": [\"7584133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CD28 costimulation enhances T cell survival during activation by upregulating Bcl-xL expression; this survival function is independent of Fas expression and cell death in the absence of CD28 costimulation displays apoptotic morphology reversible by ICE protease inhibitors\",\n      \"method\": \"CD28 KO mouse T cell viability assays, CTLA4Ig treatment, Bcl-xL/Bcl-2 expression analysis, ICE protease inhibitor treatment\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO plus pharmacological inhibition with multiple orthogonal readouts\",\n      \"pmids\": [\"8752911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cbl-b deficiency abolishes the CD28 dependence of T cell activation for IL-2 production; the Cbl-b-null mutation fully restores T cell-dependent antibody responses in CD28-/- mice; CD28 signals through Cbl-b to regulate Vav (guanine nucleotide exchange factor for Rac1/Rho/CDC42) activation\",\n      \"method\": \"Cbl-b knockout mouse generation, IL-2 production assays, genetic epistasis with CD28-/- mice, Vav activation analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double KO plus mechanistic pathway identification\",\n      \"pmids\": [\"10646609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A Y170F point mutation in mouse CD28 (Y173F in human) uncouples SH2-mediated interactions (PI3K, Grb2, Gads) from CD28 signaling; this mutant cannot upregulate Bcl-xL and sensitizes T cells to radiation-induced death, but still prevents anergy and promotes T cell proliferation, IL-2 secretion and B cell help — separating survival signals from proliferative signals\",\n      \"method\": \"Transgenic knockin mice expressing CD28 Y170F mutant, T cell activation and apoptosis assays, Bcl-xL expression, IL-2 production\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vivo mutagenesis with multiple orthogonal functional readouts dissecting distinct signaling outputs\",\n      \"pmids\": [\"11276203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HIV-1 and SIV Nef proteins down-modulate CD28 surface expression by accelerating CD28 endocytosis via the AP-2 clathrin adaptor; CD28 co-localizes with AP-2 and Nef-GFP; mutations disrupting Nef-AP-2 interaction disrupt CD28 down-regulation; Nef forms a ternary complex with AP-2 and CD28 via membrane-proximal cytoplasmic domain of CD28\",\n      \"method\": \"Nef-GFP co-localization, AP-2 mutation analysis, CD28 endocytosis assays, genetic dissection of Nef binding sites\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including mutagenesis, co-localization, and functional endocytosis assay\",\n      \"pmids\": [\"11285224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD28 enhances T cell/APC close contacts and amplifies PLCgamma1 activation and Ca2+ response without increasing Zap-70 and Lat phosphorylation; the PTK Itk controls PLCgamma1 amplification downstream of CD28; CD28 contributes to setting phosphorylated Lat levels for signaling complex recruitment\",\n      \"method\": \"Live imaging, phosphoprotein analysis, PLCgamma1 activation assays, Itk-deficient cell comparisons\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical approaches with genetic validation\",\n      \"pmids\": [\"11754815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD4 recruits Lck to the T cell-APC interface while CD28 sustains Lck autophosphorylation/activation; TCR cross-linking alone cannot stimulate Lck autophosphorylation; CD45 is not required for this process\",\n      \"method\": \"Phosphospecific antiserum to Lck, T cell stimulation by APCs, genetic ablation of CD4/CD28\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — phosphospecific reagents with genetic dissection in defined cellular context\",\n      \"pmids\": [\"11828322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TNF-alpha reduces CD28 cell surface expression by inhibiting the CD28 minimal promoter transcriptional activity; TNF-alpha inactivates promoter through reduction of DNA-protein complex formation at initiator motifs (site alpha and beta); nuclear extracts from TNF-alpha-treated cells failed to activate transcription from CD28 initiator sequences in vitro transcription assays\",\n      \"method\": \"Reporter gene assays, in vitro transcription assays, EMSA, flow cytometry\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro transcription reconstitution plus promoter binding assays with functional validation\",\n      \"pmids\": [\"11544310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CD28 costimulation, acting through PI3K and Akt, is required for T cells to increase glycolytic rate and glucose uptake in response to activation; CD28 induces glucose uptake and glycolysis in excess of that needed to maintain ATP/ADP levels, anticipating biosynthetic needs\",\n      \"method\": \"Glucose uptake assays, glycolysis measurements, PI3K/Akt inhibitor studies, CD28 KO T cells\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO cells plus pharmacological pathway dissection with multiple metabolic readouts\",\n      \"pmids\": [\"12121659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD28 signals via CD80 and CD86 to dendritic cells in reverse (bidirectional signaling): soluble CD28 induces mouse dendritic cells to express IL-6 and IFN-gamma; IL-6 production requires B7-1, B7-2, and p38 MAPK; this prevents IFN-gamma-driven tryptophan catabolism and acts as an adjuvant in vivo\",\n      \"method\": \"Soluble CD28 treatment of dendritic cells, cytokine ELISA, p38 MAPK inhibitor, B7-1/B7-2 blocking antibodies, in vivo immunization models\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches including genetic and pharmacological dissection with in vivo validation\",\n      \"pmids\": [\"15467723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of soluble CD28 in complex with a mitogenic antibody Fab fragment defines CD28 as a monovalent homodimer; structural comparisons explain distinct ligand-binding and stoichiometric properties of CD28 versus CTLA-4; cryo-EM of CD28 with mitogenic vs. non-mitogenic antibodies places constraints on receptor triggering models\",\n      \"method\": \"X-ray crystallography, cryo-electron microscopy\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with cryo-EM validation\",\n      \"pmids\": [\"15696168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD28 costimulation induces stable epigenetic modification of the IL-2 promoter: CD28 signals drive histone acetylation, cytosine demethylation, and chromatin remodeling at the IL-2 locus; TCR activation without CD28 fails to promote these changes; anergic cells maintain inaccessible IL-2 promoter chromatin\",\n      \"method\": \"Chromatin accessibility assays, histone acetylation analysis, bisulfite sequencing for cytosine methylation, CD4+ T cell anergy model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal epigenetic methods comparing CD28-stimulated vs. anergic cells\",\n      \"pmids\": [\"15814687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD28 engagement induces protein arginine methyltransferase activity and arginine methylation of several proteins including Vav1; methylated Vav1 localizes selectively to the nucleus; inhibiting S-adenosyl-L-homocysteine hydrolase reduces Vav1 methylation and IL-2 production\",\n      \"method\": \"In vitro methyltransferase assays, subcellular fractionation, immunoprecipitation, pharmacological inhibition of transmethylation\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro biochemical assay plus cellular pharmacological and fractionation studies\",\n      \"pmids\": [\"16061726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD28 signaling involves PI3K (via YMNM motif), Grb2, Filamin A, PKCtheta, and phosphatases; CTLA-4 activates PI3K and PKB/AKT to ensure cell survival under anergy-induction conditions; CD28 and CTLA-4 form signaling complexes with distinct downstream effectors controlling T cell immunity\",\n      \"method\": \"Co-immunoprecipitation, signaling pathway analysis, phosphoprotein detection\",\n      \"journal\": \"Immunological reviews\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — review consolidating biochemical findings from multiple studies, single-method evidence for some interactions\",\n      \"pmids\": [\"19426212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p85beta (PI3K regulatory subunit) binds CD28 with greater affinity than p85alpha and also binds CBL; deletion of p85beta impairs CD28-induced c-CBL and CBL-b down-regulation and PI3K pathway activation, resulting in defective T cell differentiation and failed secondary immune responses\",\n      \"method\": \"p85beta knockout mice, co-immunoprecipitation, PI3K activity assays, T cell recall response assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice combined with biochemical binding studies and functional immune response assays\",\n      \"pmids\": [\"19190244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"B7-H2 (ICOSL) is a ligand for CD28 and CTLA-4 in humans (but not mice); B7-H2-CD28 interaction costimulates human T cell primary and memory responses; B7-H2-CD28 engagement induces Bcl-xL, downregulates p27kip1, and triggers ERK and AKT signaling cascades\",\n      \"method\": \"Receptor array assay, binding assays with distinct domain mutants, T cell stimulation assays, signaling pathway analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — receptor array plus multiple orthogonal functional assays with domain mapping\",\n      \"pmids\": [\"21530327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD28 has a cell-intrinsic obligate post-maturational role in Treg function; Treg-specific CD28 conditional KO mice develop severe autoimmunity with activated T cell accumulation, dampened CTLA-4, PD-1, and CCR6 expression on Tregs, and Treg proliferative/survival disadvantage in competitive environments\",\n      \"method\": \"Treg-specific conditional Cd28 KO (Cre-lox), adoptive transfer complementation, flow cytometry, autoimmune disease models\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with multiple phenotypic readouts and competitive complementation experiments\",\n      \"pmids\": [\"23281398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD28 and CD3/TCR provide complementary functions in T cell traction force generation: CD3 mediates force generation while CD28 engagement increases traction forces through PI3K signaling; forces are concentrated at the cell periphery in complexes containing phosphorylated Pyk2\",\n      \"method\": \"Traction force microscopy on elastomer pillar arrays, PI3K inhibitor studies, micropatterned antibody presentation, primary human T cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biophysical reconstitution with pharmacological pathway dissection and defined spatial readouts\",\n      \"pmids\": [\"24469820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CTLA4-CD28 gene fusions (encoding CTLA4 extracellular domain fused to CD28 cytoplasmic domain) are found in 58% of angioimmunoblastic T-cell lymphomas; ectopic expression of the fusion in Jurkat and H9 cells enhances proliferation and AKT/ERK phosphorylation, converting inhibitory signals to stimulatory oncogenic signals\",\n      \"method\": \"RNA sequencing, RT-PCR/Sanger sequencing of 115 T-cell lymphoma samples, ectopic expression in Jurkat/H9 cells, AKT/ERK phosphorylation assays\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional expression study with signaling readouts, large clinical validation cohort\",\n      \"pmids\": [\"26819049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Bacterial superantigens bind both CD28 and its coligand B7-2 simultaneously at their homodimer interfaces (using a 12-aa beta-strand-hinge-alpha-helix domain), enhancing avidity between B7-2 and CD28 to induce T cell hyperactivation and cytokine storm; B7-2 dimer interface mimetic peptides block superantigen binding and prevent lethality in mice\",\n      \"method\": \"Superantigen binding assays to B7-2 and CD28, murine protection experiments, B7-2/CD28 avidity measurements, peptide inhibitor studies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple binding assays plus in vivo mouse protection model\",\n      \"pmids\": [\"27708164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD28 transiently promotes expression of Cpt1a (facilitating mitochondrial FAO) before the first cell division upon T cell activation; miR-33 (a target of TXNIP) attenuates Cpt1a in the absence of CD28; early CD28 signals enable T cells to remodel cristae, develop spare respiratory capacity, and produce cytokines upon restimulation — required for protective memory T cell generation\",\n      \"method\": \"Metabolic flux analysis, mitochondrial imaging, miR-33 manipulation, TXNIP studies, CD28 KO T cells, gene expression profiling\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal metabolic and molecular methods with KO comparison\",\n      \"pmids\": [\"28919076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD28 conformation and signaling are regulated by acidic phospholipids (sequestering CD28 signaling motifs within the membrane, limiting basal signaling) and Ca2+ ions (disrupting CD28-lipid interaction, leading to CD28 opening and signaling); TCR activation raises local Ca2+ concentration around CD28; TCR, Ca2+, and CD28 form a dual-positive-feedback circuit amplifying T cell signaling\",\n      \"method\": \"NMR spectroscopy, Ca2+ concentration measurements, lipid-binding assays, T cell signaling readouts\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural analysis combined with functional signaling validation\",\n      \"pmids\": [\"29058713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD28 transduces pro-survival signaling in long-lived plasma cells (LLPCs) through differential SLP76 expression; CD28 signaling in LLPCs increases glucose uptake, mitochondrial mass/respiration, and ROS production; CD28-mediated mitochondrial respiration, NF-κB activation, and survival are ROS-dependent; IRF4 (NF-κB target) is upregulated by CD28 and mediates these metabolic effects\",\n      \"method\": \"LLPC vs SLPC CD28 signaling comparisons, metabolic flux analysis, ROS measurement, NF-κB reporter assays, IRF4 knockdown\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal metabolic and signaling assays in primary cells with mechanistic pathway dissection\",\n      \"pmids\": [\"32579940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The CD28 transmembrane domain (TMD) mediates heterodimerization of CD28-containing CARs with endogenous CD28 in human T cells; this dimerization depends on polar amino acids in the CD28-TMD; CD28-TMD CARs show CAR-dependent proliferation upon anti-CD28 stimulation but do not respond to CD80/CD86 and reduce CD28 surface expression\",\n      \"method\": \"Co-immunoprecipitation, anti-CD28 stimulation assays, polar amino acid mutagenesis, flow cytometry for CD28 surface expression\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP with mutagenesis and functional validation in primary human T cells\",\n      \"pmids\": [\"33833759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CARMIL2 is required for canonical NF-κB (but not AP-1 and NFAT) activation in T cells stimulated via CD28; CARMIL2-deficient patients share features of CD28 deficiency (low memory T cells, recalcitrant warts) but additionally show low NK cells, memory B cells, and weak antibody responses; somatic reversions in CD4+ T cells associate with milder phenotypes\",\n      \"method\": \"Functional complementation assays with mutant CARMIL2 alleles, NF-κB/AP-1/NFAT reporter assays, patient immunophenotyping, somatic reversion analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional pathway dissection with multiple isoforms and patient-derived cells, genetic reversion evidence\",\n      \"pmids\": [\"36515678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CD8+ T cells display B7 ligands (CD80/CD86) that interact with CD28 in cis at membrane invaginations of the immunological synapse; cis-B7:CD28 interactions are driven by PI3K and sorting-nexin-9 (SNX9)-mediated membrane remodeling; cis-CD28 signaling occurs through PKCθ and promotes CD8+ T cell survival, migration, and cytokine production; loss of T cell-intrinsic cis-B7:CD28 interactions decreases intratumoral T cells and accelerates tumor growth\",\n      \"method\": \"Live imaging, PI3K/SNX9 knockout/inhibitor studies, PKCθ signaling assays, in vivo mouse tumor models\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches including imaging, genetic KO, pharmacological, and in vivo tumor models\",\n      \"pmids\": [\"37160118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CD28 incorporated into CAR constructs in NK cells recruits LCK and ZAP70 kinases to the CD3ζ signaling axis, initiating a signaling cascade that enhances CAR-NK cell function; CD28 is not inherently present in mature NK cells but its introduction significantly enhances antitumor efficacy and long-term cytotoxicity in vitro and in xenograft models\",\n      \"method\": \"Co-immunoprecipitation of LCK/ZAP70 with CD28-CD3ζ, kinase recruitment assays, in vitro cytotoxicity, xenograft mouse models\",\n      \"journal\": \"Cancer discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical recruitment assays plus multiple in vivo models\",\n      \"pmids\": [\"38900051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CD2 and CD28 function together to facilitate T cell-APC interactions enabling TCR signal transduction; mice lacking both CD2 and CD28 have profound defects in T cell activation by soluble anti-CD3 and antigen, while remaining responsive to immobilized anti-CD3\",\n      \"method\": \"CD2/CD28 double-knockout mouse generation, T cell activation assays with soluble vs. immobilized anti-CD3\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double KO revealing cooperative function\",\n      \"pmids\": [\"10725714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PI3K associated with the CD28 YMNM motif acts as a negative mediator in CD28-mediated IL-2 gene transcription; Y189F mutant (disrupting PI3K binding) shows reduced but significant IL-2 promoter activity, while N191A mutant (retains PI3K binding) completely abrogates activity; PI3K inhibitor augments IL-2 promoter activity while constitutively active PI3K reduces it\",\n      \"method\": \"CD28 YMNM point and deletion mutants expressed in Jurkat cells, IL-2 promoter reporter assays, PI3K pharmacological inhibitors, constitutively active PI3K expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis plus pharmacological gain/loss of function with reporter gene readout\",\n      \"pmids\": [\"11113113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Itk kinase negatively regulates CD28-mediated T cell proliferation; Itk-deficient T cells show significantly elevated proliferative response to CD28 costimulation compared to controls, while CD3-mediated responses are severely compromised; Itk differentially modulates CD3 vs. CD28 signaling pathways\",\n      \"method\": \"Itk knockout mouse T cell stimulation assays comparing CD28 vs. CD3 pathway responses\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with pathway-specific dissection\",\n      \"pmids\": [\"9221751\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD28 is a homodimeric costimulatory receptor on T cells that binds B7-1 (CD80), B7-2 (CD86), and B7-H2 (in humans) on APCs; upon ligation its cytoplasmic YMNM motif becomes tyrosine-phosphorylated, recruiting PI3K (p85alpha/p85beta), Grb2, and Gads to activate Akt-driven glucose metabolism, Vav-driven actin remodeling, PLCgamma1-driven Ca2+ and PKCθ signals, NF-κB via CARMIL2, and Bcl-xL-mediated survival; CD28 also epigenetically remodels the IL-2 locus, primes mitochondrial fatty acid oxidation capacity before the first cell division to enable memory formation, sustains Lck activation at the immunological synapse, and can signal in cis via T cell-displayed B7 ligands through PI3K/SNX9-driven membrane remodeling.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1987,\n      \"finding\": \"CD28 (Tp44) is a T-cell-specific homodimeric surface protein with homology to the immunoglobulin superfamily; its cDNA was cloned by transient COS cell expression, revealing it directs production of a disulfide-bonded homodimer.\",\n      \"method\": \"COS cell expression cloning, monoclonal antibody binding, molecular characterization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original molecular cloning with functional protein production confirmed in transfected cells\",\n      \"pmids\": [\"2825196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"CD28 (9.3 antigen) is a disulfide-bonded dimer of a 44 kDa polypeptide that defines a novel T cell activation pathway independent of the T3/Ti complex, inducing IL-2 receptor expression and IL-2 secretion when crosslinked with TPA.\",\n      \"method\": \"Monoclonal antibody precipitation, co-modulation experiments, T cell activation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — original functional characterization with multiple orthogonal methods in primary T cells\",\n      \"pmids\": [\"3159820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"CD28 is the primary receptor for B7 on T cells; B7-CD28 interaction (direct binding, Kd ~200 nM) costimulates T cell proliferation and specifically increases IL-2 mRNA accumulation.\",\n      \"method\": \"Ig fusion protein binding assay, B7-transfected CHO cell costimulation, IL-2 transcript measurement\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct binding reconstitution with Kd measurement, functional validation with transfected cells\",\n      \"pmids\": [\"1847722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"B7 ligation of CD28 on T cells induces proliferation and high-level IL-2 secretion; this response is specific and abrogated by anti-B7 antibody.\",\n      \"method\": \"B7-transfected CHO cell costimulation assay, antibody blockade, cytokine measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated functional finding with specific blocking controls\",\n      \"pmids\": [\"1650475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"B70 (B7-2/CD86) is a second ligand for CD28 and CTLA-4; monoclonal antibody to B70 inhibits CTLA4-Ig binding to B lymphoblastoid cells and blocks primary allogeneic MLR.\",\n      \"method\": \"cDNA cloning, CTLA4-Ig fusion protein binding inhibition, mixed lymphocyte reaction\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ligand identified by cDNA cloning, binding competition, and functional blockade\",\n      \"pmids\": [\"7694153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"CD28-knockout mice have impaired lectin-stimulated IL-2 production and reduced IL-2 receptor alpha expression; CD28 is required for T helper cell activity and immunoglobulin class switching but not for all T cell responses, indicating alternative costimulatory pathways exist.\",\n      \"method\": \"Genetic knockout mouse, T cell activation assays, immunoglobulin measurement, infection models\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with multiple defined phenotypic readouts\",\n      \"pmids\": [\"7688139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"CD28 receptor ligation induces tyrosine phosphorylation of specific substrates including phospholipase C gamma 1, and triggers both calcium-dependent and calcium-independent signals.\",\n      \"method\": \"Phosphorylation assays in activated T cells\",\n      \"journal\": \"Annual review of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — biochemical identification of phosphorylated substrates, single method\",\n      \"pmids\": [\"8386518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"PI3K (phosphatidylinositol 3-kinase) binds directly to the phosphorylated YMNM (Y191MXM) motif in the CD28 cytoplasmic tail via the SH2 domains of the p85 subunit; mutation of Y191 abolishes PI3K binding and CD28-mediated IL-2 production.\",\n      \"method\": \"Site-directed mutagenesis, baculoviral p85 reconstitution, lipid kinase assay, HPLC, peptide competition\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct binding reconstituted in vitro with mutagenesis and multiple biochemical assays\",\n      \"pmids\": [\"8146197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CD28 becomes tyrosine-phosphorylated after B7.1/CD80 engagement, inducing complex formation with PI3K via the p85 SH2 domain at the Y173 (YXXM) motif; CD28 mutants lacking this site cannot stimulate IL-2 production.\",\n      \"method\": \"Tyrosine phosphorylation assay, co-immunoprecipitation, CD28 mutagenesis, IL-2 production assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with direct biochemical binding and functional readout, independently replicated\",\n      \"pmids\": [\"8183372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CD28 associates with PI3K following CD28 cross-linking, and a synthetic peptide representing the YMNM motif from the CD28 cytoplasmic tail interacts with PI3K only when the tyrosine is phosphorylated.\",\n      \"method\": \"Co-immunoprecipitation in Jurkat cells, phosphopeptide binding assay\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with phosphopeptide confirmation, single lab\",\n      \"pmids\": [\"8080844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The CD28 pYMNM motif Y191 mutation disrupts both PI3K and Grb2 binding; M194 mutation disrupts only PI3K binding; both mutants fail to support IL-2 production upon B7-2 (CD86) engagement, directly implicating PI3K in CD28-mediated costimulation.\",\n      \"method\": \"CD28 point mutant expression, PI3K and Grb2 binding assays, IL-2 production assay with B7-2 transfected cells\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — selective mutagenesis dissecting binding partners with functional readout\",\n      \"pmids\": [\"7584133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CD28 costimulation upregulates Bcl-xL expression to enhance T cell survival during activation; CD28-deficient T cells show increased apoptosis that can be blocked by ICE protease inhibitors; this survival function is Fas-independent.\",\n      \"method\": \"CD28-KO mouse T cells, Bcl-xL/Bcl-2/Fas protein expression, apoptosis assays, lpr mouse comparison, ICE protease inhibitor treatment\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple orthogonal methods and specific molecular mechanism identified\",\n      \"pmids\": [\"8752911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cbl-b, an adaptor molecule, regulates the CD28 dependence of T cell activation; Cbl-b-deficient T cells do not require CD28 engagement for IL-2 production. Cbl-b selectively suppresses TCR-mediated Vav activation, and Cbl-b loss enhances Vav (GEF for Rac1/Rho/CDC42) activation.\",\n      \"method\": \"Cbl-b knockout mice, IL-2 production assay, genetic epistasis with CD28-KO mice, Vav activation assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double-KO rescue and defined molecular pathway\",\n      \"pmids\": [\"10646609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD28 costimulation prevents anergy and promotes T cell proliferation via a PI3K-independent pathway; a Y170F point mutation in CD28 uncouples SH2-dependent signaling (PI3K/Grb2/Gads) from CD28, abolishing Bcl-xL upregulation and survival but preserving anergy prevention, proliferation, IL-2 secretion, and B cell help.\",\n      \"method\": \"Transgenic knockin mouse expressing CD28-Y170F mutant, T cell activation assays, Bcl-xL expression, anergy assessment\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockin mouse with point mutation separating distinct signaling outputs\",\n      \"pmids\": [\"11276203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SIV and HIV-1 Nef proteins downmodulate CD28 by accelerating its endocytosis via the AP-2 clathrin adaptor pathway; Nef, AP-2, and CD28 form a ternary complex involving the membrane-proximal CD28 cytoplasmic domain, and Nef mutations disrupting AP-2 interaction abrogate CD28 downregulation.\",\n      \"method\": \"Nef-GFP fusion protein co-localization, co-immunoprecipitation with AP-2, endocytosis assay, Nef mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic mutagenesis combined with co-localization and co-IP evidence\",\n      \"pmids\": [\"11285224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD28 amplifies TCR signaling by enhancing T cell–APC contacts and specifically boosting PLCgamma1 activation and Ca2+ response downstream of TCR, without increasing Zap-70 or LAT phosphorylation; the PTK Itk controls the PLCgamma1 amplification function.\",\n      \"method\": \"Biochemical analysis of signaling intermediates (phospho-Zap70, phospho-LAT, PLCgamma1 activation, Ca2+ flux), Itk involvement assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical readouts defining mechanism\",\n      \"pmids\": [\"11754815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TNF-alpha directly suppresses CD28 gene transcription by inhibiting activity of the CD28 minimal promoter through reduction of DNA-protein complex formation at initiator sequence motifs (sites alpha and beta), as shown by reporter gene assays and in vitro transcription assays.\",\n      \"method\": \"Reporter gene bioassay, in vitro transcription assay, EMSA (DNA-protein complex formation), nuclear extract analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple complementary in vitro methods with molecular mechanism at promoter level\",\n      \"pmids\": [\"11544310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CD28 signaling through PI3K and Akt is required for T cells to increase glycolytic rate and glucose uptake upon activation; CD28 costimulation drives glycolytic flux in excess of immediate ATP/ADP needs, preparing cells for sustained metabolic demands.\",\n      \"method\": \"Glucose uptake assays, glycolysis measurements, PI3K/Akt inhibitor treatment, CD28-deficient T cell comparison\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple metabolic assays with genetic and pharmacological perturbation of PI3K/Akt pathway\",\n      \"pmids\": [\"12121659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Lck undergoes autophosphorylation in the immunological synapse when T cells encounter APCs; CD4 recruits Lck to the T cell–APC interface, whereas CD28 sustains Lck activation; TCR cross-linking alone cannot stimulate Lck autophosphorylation.\",\n      \"method\": \"Phosphospecific anti-Lck antiserum, APC stimulation, CD4 and CD28 perturbation, immunofluorescence\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — phosphospecific reagents with mechanistic dissection of receptor contributions\",\n      \"pmids\": [\"11828322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD28 co-localizes with protein kinase C theta (PKCtheta) within TCR-CD28 microclusters at the immunological synapse, and this association promotes sustained T cell signaling from a specialized subregion of the IS after mature synapse formation.\",\n      \"method\": \"Live cell imaging, microcluster analysis, immunological synapse imaging\",\n      \"journal\": \"Immunological reviews\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization with functional implication, single lab review synthesis\",\n      \"pmids\": [\"19426213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Soluble CD28 signals through B7-1 (CD80) and B7-2 (CD86) on dendritic cells to induce IL-6 and IFN-gamma production; IL-6 production requires CD80, CD86, and p38 MAPK and prevents IFN-gamma-driven immunosuppressive tryptophan catabolism, demonstrating reverse (inside-out) B7 signaling.\",\n      \"method\": \"Soluble CD28 treatment of DCs, cytokine measurement, p38 MAPK inhibition, in vivo adjuvant assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including pharmacological inhibition and in vivo validation\",\n      \"pmids\": [\"15467723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of soluble CD28 in complex with a mitogenic antibody Fab fragment reveals CD28 is a monovalent homodimer, redefining evolutionary relationships within the CD28/CTLA-4 superfamily and accounting for distinct ligand-binding and stoichiometric properties versus CTLA-4; cryo-EM comparisons of mitogenic vs. non-mitogenic antibody complexes constrain receptor-triggering models.\",\n      \"method\": \"X-ray crystallography, cryo-electron microscopy\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus cryo-EM, structurally validated functional differences\",\n      \"pmids\": [\"15696168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"B7-1 and B7-2 differ in affinity and structure: B7-2 binds CD28 and CTLA-4 more weakly than B7-1; relative to CTLA-4 binding, B7-2 binds CD28 2–3-fold more effectively than B7-1; B7-1 is bivalent (self-associates) while B7-2 does not; CD28 homodimers are monovalent whereas CTLA-4 homodimers are bivalent.\",\n      \"method\": \"Surface plasmon resonance, analytical ultracentrifugation, structural comparison\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biophysical binding measurements with structural validation\",\n      \"pmids\": [\"12196291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD28 costimulation induces stable epigenetic modification of the IL-2 locus: histone acetylation, cytosine demethylation, and chromatin remodeling to an accessible state; TCR activation without CD28 fails to promote these epigenetic changes, leaving the IL-2 promoter inaccessible in anergic cells.\",\n      \"method\": \"Chromatin accessibility assay, bisulfite sequencing (cytosine methylation), ChIP (histone acetylation), comparison of activated vs. anergic T cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal epigenetic assays with clean anergy comparison\",\n      \"pmids\": [\"15814687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD28 engagement induces protein arginine methyltransferase activity and arginine methylation of several proteins including Vav1; methylated Vav1 localizes selectively to the nucleus; inhibiting transmethylation reduces Vav1 methylation and IL-2 production.\",\n      \"method\": \"Protein arginine methyltransferase activity assay, arginine methylation detection, subcellular fractionation, pharmacological inhibition of S-adenosyl-L-homocysteine hydrolase\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — novel PTM identified with mechanistic inhibitor and localization data\",\n      \"pmids\": [\"16061726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p85beta (regulatory subunit of PI3K) binds to CD28 and to CBL with greater affinity than p85alpha; deletion of p85beta impairs CD28-induced c-CBL and CBL-b downregulation and PI3K pathway activation, resulting in defective T cell differentiation and failure of secondary immune response.\",\n      \"method\": \"p85beta-KO mice, co-immunoprecipitation, PI3K activation assay, CBL expression measurement, secondary immune response assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse combined with co-IP and functional assays\",\n      \"pmids\": [\"19190244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Genomic analysis shows CD28 agonists alone induce few gene expression changes; the principal effect of CD28 costimulation is amplification of the CD3 transcriptional response; CD28 promotes phosphorylation/inactivation of GSK3 (NFAT nuclear export kinase), enhancing nuclear NFAT accumulation and NFAT target gene expression.\",\n      \"method\": \"cDNA microarray, GSK3 phosphorylation assay, NFAT dephosphorylation assay, FK506 (calcineurin inhibitor) treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — microarray plus biochemical pathway validation with pharmacological controls\",\n      \"pmids\": [\"12195013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"B7-H2 (ICOSL) functions as a ligand for CD28 and CTLA-4 in humans (but not mice); B7-H2–CD28 interaction uses a distinct binding domain from B7-1/B7-2; B7-H2 costimulation via CD28 induces Bcl-xL, downregulates p27(kip1), and activates ERK and AKT signaling pathways.\",\n      \"method\": \"Receptor array assay, binding domain mapping, T cell costimulation assays, signaling pathway analysis (ERK, AKT phosphorylation), Bcl-xL and p27 expression\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ligand discovery by receptor array with multiple orthogonal functional validations\",\n      \"pmids\": [\"21530327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD28 has an obligate cell-intrinsic function in Tregs beyond thymic development; Treg-specific Cd28 conditional knockout mice develop severe autoimmunity despite normal FOXP3+ cell numbers; CD28 loss in Tregs dampens CTLA-4, PD-1, and CCR6 expression and confers a proliferative/survival disadvantage in competitive environments.\",\n      \"method\": \"Treg-specific conditional CD28 knockout mice, flow cytometry, in vivo autoimmune models (EAE), Treg complementation experiments\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with multiple phenotypic readouts and rescue experiments\",\n      \"pmids\": [\"23281398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD28 and CD3 provide complementary functions in T cell force generation: force generation is associated with CD3/TCR complex engagement, while CD28 increases traction forces associated with CD3 through the PI3K signaling pathway rather than by direct cell-substrate coupling; phosphorylated Pyk2 is concentrated at sites of force generation.\",\n      \"method\": \"Traction force microscopy on elastomer pillar arrays, PI3K inhibitor treatment, phospho-Pyk2 localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — quantitative biophysical assay with pharmacological pathway dissection\",\n      \"pmids\": [\"24469820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Bacterial superantigens bind both B7-2 and CD28 at their homodimer interfaces using the same 12-aa domain, potently enhancing B7-2/CD28 avidity and inducing T cell hyperactivation; short B7-2 dimer interface mimetic peptides prevent superantigen binding and protect mice from lethal challenge.\",\n      \"method\": \"Binding assays, B7-2 and CD28 homodimer interface mutagenesis, cytokine production assays, mouse lethality model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic mutagenesis with in vivo validation\",\n      \"pmids\": [\"27708164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD28 conformation and signaling are regulated by two counteractive charged factors: acidic phospholipids sequester CD28 cytoplasmic signaling motifs within the membrane (limiting basal signaling), while TCR-induced local Ca2+ increase directly disrupts CD28-lipid interaction, releasing the cytoplasmic domain for signaling; TCR, Ca2+, and CD28 form a dual-positive-feedback circuit amplifying T cell signaling.\",\n      \"method\": \"NMR spectroscopy of CD28 transmembrane/cytoplasmic domain, lipid binding assays, Ca2+ perturbation experiments, T cell signaling assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with mechanistic functional validation\",\n      \"pmids\": [\"29058713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD28 transiently promotes expression of CPT1a (carnitine palmitoyltransferase 1a) before the first cell division, facilitating mitochondrial fatty acid oxidation, cristae remodeling, and enhanced spare respiratory capacity; miR-33 (targeted by TXNIP) attenuates Cpt1a in the absence of CD28; early CD28-dependent mitochondrial priming is essential for memory T cell formation.\",\n      \"method\": \"CD28-deficient T cells, Cpt1a expression measurement, miR-33 and TXNIP analysis, mitochondrial imaging, spare respiratory capacity assay, cytokine production upon restimulation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal metabolic and molecular assays in KO context with temporal resolution\",\n      \"pmids\": [\"28919076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CTLA4-CD28 gene fusion (encoding CTLA4 extracellular domain fused to CD28 cytoplasmic domain) is frequently found in angioimmunoblastic T-cell lymphoma (58%), PTCL-NOS (23%), and extranodal NK/T-cell lymphoma (29%); ectopic expression in Jurkat/H9 cells enhances proliferation and activates AKT and ERK downstream of what would normally be an inhibitory CTLA4 signal.\",\n      \"method\": \"RNA sequencing, RT-PCR/Sanger sequencing, ectopic expression in T cell lines, AKT/ERK phosphorylation assay\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional validation of gene fusion in multiple cell lines with signaling readout\",\n      \"pmids\": [\"26819049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CARs containing the CD28 transmembrane domain (TMD) heterodimerize with endogenous CD28 in human T cells via polar amino acids in the CD28-TMD; this heterodimerization reduces CD28 cell-surface expression and does not respond to CD80/CD86 stimulation, revealing a fundamental functional difference from CD8-TMD CARs.\",\n      \"method\": \"Co-immunoprecipitation, CAR-dependent proliferation assay upon anti-CD28 stimulation, CD28-TMD mutagenesis, flow cytometry\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with mutagenesis and functional validation\",\n      \"pmids\": [\"33833759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CD8+ T cells display B7 ligands (CD80/CD86) that interact with CD28 in cis at invaginated synaptic membranes; this cis-B7:CD28 interaction is driven by PI3K and sorting nexin-9 (SNX9), triggers CD28 signaling through PKCtheta, and promotes CD8+ T cell survival, migration, and cytokine production; loss of cis-B7:CD28 interactions in mouse tumor models decreases intratumoral T cells and accelerates tumor growth.\",\n      \"method\": \"Live cell imaging, co-IP, PI3K and SNX9 perturbation, PKCtheta signaling assay, mouse tumor models with T cell-specific B7 knockout\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection with multiple orthogonal methods and in vivo validation\",\n      \"pmids\": [\"37160118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD28 transduces pro-survival signaling in long-lived plasma cells (LLPCs) specifically through differential SLP76 expression; CD28 signaling in LLPCs increases glucose uptake, mitochondrial mass/respiration, and ROS production; CD28-mediated NF-κB activation and survival are ROS-dependent; IRF4 (NF-κB target) upregulated by CD28 mediates this metabolic program.\",\n      \"method\": \"LLPC vs. SLPC comparison, SLP76 expression analysis, glucose uptake, mitochondrial assays, ROS measurement, NF-κB activation assay, IRF4 expression\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal metabolic and signaling assays with mechanistic dissection\",\n      \"pmids\": [\"32579940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CD28 costimulatory domain incorporated into CAR-NK cells recruits LCK and ZAP70 kinases to the CD3ζ signaling complex, initiating a signaling cascade that enhances CAR-NK cell antitumor persistence and cytotoxicity.\",\n      \"method\": \"CAR-NK cell engineering, kinase recruitment assay, in vitro cytotoxicity assay, multiple xenograft mouse models\",\n      \"journal\": \"Cancer discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic kinase recruitment demonstrated with in vitro and in vivo validation\",\n      \"pmids\": [\"38900051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Itk negatively regulates CD28-mediated T cell proliferation: Itk-deficient T cells show augmented proliferative response to CD28 costimulation (calcineurin-independent) without increased IL-2, establishing that Itk plays distinct inhibitory roles in CD28 versus CD3 signaling pathways.\",\n      \"method\": \"Itk-knockout mice, T cell proliferation assay, IL-2 measurement, calcineurin pathway inhibition\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with defined pathway dissection\",\n      \"pmids\": [\"9221751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CD28 can activate PI3K and cooperate with adapters Vav and SLP-76 to induce IL-2 and IL-4 transcription independently of TCR ligation, providing a molecular basis for in trans costimulation.\",\n      \"method\": \"CD28 stimulation without TCR ligation, IL-2/IL-4 promoter assays, Vav and SLP-76 co-signaling analysis\",\n      \"journal\": \"Immunological reviews\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pathway characterization, single method per component\",\n      \"pmids\": [\"12670393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Unexpectedly, the Y189F CD28 YMNM mutant that disrupts PI3K binding shows reduced but significant IL-2 promoter activity, while N191A (retains PI3K binding) completely abrogates activity; PI3K inhibitor augments IL-2 promoter activity while constitutively active PI3K reduces it, suggesting PI3K acts as a negative mediator of CD28-mediated IL-2 transcription.\",\n      \"method\": \"CD28 YMNM point/deletion mutant expression in Jurkat, IL-2 promoter reporter assay, PI3K pharmacological inhibitor, constitutively active PI3K overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple mutants and pharmacological tools, but finding contradicts other studies (context-dependent)\",\n      \"pmids\": [\"11113113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD28 co-stimulatory pathway, specifically through the Tec-family kinase ITK, regulates trafficking of autoreactive T cells to tissues; ablation of ITK in Ctla4−/− mice blocks tissue infiltration without preventing lymphoid organ T cell activation; ITK inhibitors phenocopy ITK null and prevent islet infiltration in diabetes models.\",\n      \"method\": \"Double-KO mice (Itk−/−;Ctla4−/−), T cell trafficking analysis, ITK pharmacological inhibition, pancreatic islet infiltration model\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with pharmacological validation and in vivo disease model\",\n      \"pmids\": [\"24270545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CD28 costimulation drives distinct metabolic programs in CAR T cells: CD28 signaling domains favor effector memory differentiation with a glycolytic gene signature, while 4-1BB domains promote central memory with enhanced fatty acid oxidation and mitochondrial biogenesis.\",\n      \"method\": \"Metabolic flux analysis, gene expression profiling, mitochondrial assays in CAR T cells with different costimulatory domains\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple metabolic assays with direct CD28 vs. 4-1BB domain comparison\",\n      \"pmids\": [\"26885860\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD28 is a disulfide-bonded homodimeric T cell costimulatory receptor that, upon engagement by B7-1 (CD80), B7-2 (CD86), or B7-H2 (in humans), becomes tyrosine-phosphorylated at its cytoplasmic YMNM motif, recruiting PI3K (p85/p110) and adaptor proteins (Grb2, Vav, SLP-76) to activate PI3K–Akt signaling, boost PLCγ1 and Ca2+/NFAT responses, inactivate GSK3, induce arginine methylation of Vav1, upregulate Bcl-xL for survival, drive glycolysis and mitochondrial fatty acid oxidation, and promote epigenetic remodeling of the IL-2 locus—with CD28 transmembrane signaling further regulated by membrane lipid sequestration relieved by TCR-induced Ca2+, and cis-B7:CD28 interactions at invaginated synaptic membranes extending costimulation in peripheral tissues.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CD28 is a homodimeric costimulatory receptor on T cells that integrates TCR-proximal signals to drive T cell activation, survival, metabolic reprogramming, and memory formation. CD28 binds B7-1 (CD80), B7-2 (CD86), and — in humans — B7-H2 (ICOSL) on antigen-presenting cells; upon ligation, its cytoplasmic YMNM motif is tyrosine-phosphorylated and recruits PI3K, Grb2, and Gads, activating Akt-driven glucose metabolism, Vav1-mediated cytoskeletal remodeling, PLCγ1/Itk-amplified calcium signaling, PKCθ activation, NF-κB signaling via CARMIL2, Bcl-xL-dependent survival, and stable epigenetic remodeling of the IL-2 locus [PMID:8386518, PMID:8080844, PMID:7584133, PMID:8752911, PMID:15814687, PMID:36515678, PMID:12121659]. CD28 costimulation before the first cell division primes mitochondrial fatty acid oxidation capacity (via Cpt1a upregulation) required for memory T cell generation, sustains Lck activation at the immunological synapse, and can signal in cis through T cell-displayed B7 ligands via PI3K/SNX9-driven membrane remodeling to promote CD8+ T cell intratumoral survival [PMID:28919076, PMID:11828322, PMID:37160118]. CD28 also has a cell-intrinsic obligate role in regulatory T cell homeostasis, as Treg-specific CD28 deletion causes severe autoimmunity [PMID:23281398].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Identifying CD28 as the receptor for B7 ligands and its dual ligand system (B7-1 and B7-2) established the molecular basis for T cell costimulation and revealed that CD28 ligation triggers tyrosine phosphorylation of PLCγ1 and both calcium-dependent and -independent downstream signals.\",\n      \"evidence\": \"Receptor-ligand binding studies, monoclonal antibody cloning of B70/CD86, CTLA4-Ig binding inhibition, mixed lymphocyte reactions, biochemical signaling assays\",\n      \"pmids\": [\"8386518\", \"7694153\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise intracellular signaling intermediates between CD28 and PLCγ1 not yet defined\", \"Relative contributions of B7-1 vs B7-2 to distinct signaling outcomes unknown\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"CD28-deficient mice demonstrated that CD28 is essential for IL-2 production, IL-2Rα upregulation, T helper activity, and immunoglobulin class switching, establishing CD28 as a non-redundant costimulatory receptor in vivo.\",\n      \"evidence\": \"CD28 knockout mouse generation with T cell stimulation, cytokine, and immunoglobulin isotype analyses\",\n      \"pmids\": [\"7688139\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CD28 is required for all T cell subsets or only specific lineages remained unresolved\", \"CD28-independent T cell responses not mechanistically explained\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Mutagenesis of the cytoplasmic YMNM motif revealed that Y191 phosphorylation is the master switch for recruiting both PI3K and Grb2, while the M194 residue selectively controls PI3K binding — linking defined SH2-domain interactions to IL-2 production.\",\n      \"evidence\": \"Site-directed mutagenesis of CD28 cytoplasmic domain expressed with CHO-CD86 stimulator cells, IL-2 production assays\",\n      \"pmids\": [\"7584133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PI3K recruitment is positively or negatively required for IL-2 was debated\", \"Downstream effectors of Grb2 binding to CD28 not yet identified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Discovery that CD28 costimulation upregulates Bcl-xL to promote T cell survival separated the anti-apoptotic function of CD28 from its role in proliferation and cytokine production.\",\n      \"evidence\": \"CD28 KO T cell viability assays, Bcl-xL/Bcl-2 expression analysis, ICE protease inhibitor reversal of apoptosis\",\n      \"pmids\": [\"8752911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Bcl-xL induction requires PI3K specifically or other CD28-proximal signals was unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genetic epistasis showed that Cbl-b is the critical negative regulator that enforces CD28 dependence: its deletion fully restores T cell responses and antibody production in CD28-null mice, operating through Vav1 activation control.\",\n      \"evidence\": \"Cbl-b/CD28 double-knockout mice, IL-2 production, T cell-dependent antibody responses, Vav activation analysis\",\n      \"pmids\": [\"10646609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism by which CD28 relieves Cbl-b-mediated Vav ubiquitination not fully dissected\", \"Whether Cbl-b regulation of CD28 dependence operates identically in all T cell subsets unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Paradoxically, PI3K recruited to the YMNM motif was shown to negatively regulate IL-2 transcription, with the N191 residue (not Y189) being essential for positive IL-2 promoter activity — refining understanding that PI3K has context-dependent inhibitory and stimulatory roles downstream of CD28.\",\n      \"evidence\": \"YMNM point and deletion mutants in Jurkat cells, IL-2 promoter reporter assays, PI3K inhibitors, constitutively active PI3K expression\",\n      \"pmids\": [\"11113113\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PI3K simultaneously drives metabolic activation yet restrains IL-2 transcription not resolved\", \"Cell-type specificity of this negative regulatory role untested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Multiple studies in 2001 dissected distinct CD28 signaling outputs: Y170F knockin mice separated PI3K/Grb2-dependent survival (Bcl-xL) from PI3K-independent proliferation and IL-2 production; CD28 was shown to sustain Lck autophosphorylation at the synapse and amplify PLCγ1/Ca²⁺ signaling via Itk without increasing ZAP-70 or LAT phosphorylation.\",\n      \"evidence\": \"Y170F knockin mice with apoptosis/Bcl-xL/IL-2 readouts; phosphospecific Lck antibodies with APC stimulation; live imaging and PLCγ1 assays with Itk-deficient comparisons\",\n      \"pmids\": [\"11276203\", \"11828322\", \"11754815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CD28 sustains Lck activation mechanistically (direct vs indirect) not defined\", \"Structural basis for CD28-Itk cooperation unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"CD28 was established as a metabolic switch: costimulation through PI3K/Akt drives glucose uptake and glycolysis beyond immediate ATP needs, anticipating biosynthetic demands of T cell clonal expansion.\",\n      \"evidence\": \"Glucose uptake and glycolysis measurements in CD28 KO T cells with PI3K/Akt inhibitors\",\n      \"pmids\": [\"12121659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether glycolytic reprogramming is separable from survival signaling unknown\", \"Downstream metabolic targets of Akt in this context not identified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The crystal structure of CD28 revealed a monovalent homodimer with binding geometry distinct from CTLA-4, explaining differential ligand avidity and providing a structural framework for understanding costimulatory receptor triggering.\",\n      \"evidence\": \"X-ray crystallography of soluble CD28–Fab complex, cryo-EM comparison of mitogenic vs non-mitogenic antibody complexes\",\n      \"pmids\": [\"15696168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length CD28 structure including transmembrane and cytoplasmic domains not determined\", \"How monovalent binding translates to signaling initiation not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"CD28 was shown to epigenetically remodel the IL-2 locus through histone acetylation, cytosine demethylation, and chromatin opening — a function absent with TCR stimulation alone and explaining the mechanistic basis of T cell anergy when CD28 costimulation is lacking.\",\n      \"evidence\": \"Chromatin accessibility, histone acetylation, bisulfite sequencing in CD28-stimulated vs anergic CD4+ T cells\",\n      \"pmids\": [\"15814687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of chromatin-modifying enzymes recruited by CD28 signaling not established\", \"Whether epigenetic remodeling extends to loci beyond IL-2 not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of p85β as the preferred PI3K regulatory subunit for CD28 binding — with higher affinity than p85α — connected CD28 to CBL degradation control and explained why p85β deletion impairs secondary immune responses.\",\n      \"evidence\": \"p85β knockout mice, co-immunoprecipitation, PI3K activity assays, T cell recall responses\",\n      \"pmids\": [\"19190244\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p85α can partially compensate for p85β loss in specific T cell subsets unknown\", \"Structural basis of differential p85β vs p85α binding to YMNM not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Discovery that B7-H2 (ICOSL) is a third human CD28 ligand expanded the ligand repertoire beyond B7-1/B7-2, with B7-H2–CD28 engagement inducing Bcl-xL, ERK, and AKT signaling to costimulate both primary and memory T cell responses.\",\n      \"evidence\": \"Receptor array, domain-specific binding mutants, T cell stimulation with signaling pathway analysis\",\n      \"pmids\": [\"21530327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where B7-H2–CD28 interaction dominates over ICOS–B7-H2 undefined\", \"Species specificity (human only) limits translational studies in mouse models\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Treg-specific CD28 conditional deletion proved that CD28 has a cell-intrinsic, post-maturational role in regulatory T cell homeostasis: without CD28, Tregs lose CTLA-4/PD-1 expression, fail in competitive environments, and mice develop severe autoimmunity.\",\n      \"evidence\": \"Treg-specific Cre-lox CD28 conditional KO, adoptive transfer, flow cytometry, autoimmune disease phenotyping\",\n      \"pmids\": [\"23281398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise signaling pathway by which CD28 maintains Treg CTLA-4 expression not identified\", \"Whether CD28 requirement in Tregs is continuous or limited to specific activation states unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"CD28 was identified as the initiator of mitochondrial remodeling for memory T cell fate: transient Cpt1a upregulation before the first division (restrained by miR-33/TXNIP in the absence of CD28) enables cristae remodeling, spare respiratory capacity, and protective memory generation.\",\n      \"evidence\": \"Metabolic flux analysis, mitochondrial imaging, miR-33 manipulation, CD28 KO T cell comparison\",\n      \"pmids\": [\"28919076\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mitochondrial remodeling is sufficient for memory or requires additional CD28-dependent signals unknown\", \"How CD28 signaling converges on miR-33/TXNIP regulation not mechanistically defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"NMR analysis revealed that acidic phospholipids sequester CD28 cytoplasmic signaling motifs within the membrane, establishing a gating mechanism: TCR-induced local Ca²⁺ rise disrupts lipid–CD28 interactions, opening the cytoplasmic tail for SH2-domain recruitment in a dual-positive-feedback circuit.\",\n      \"evidence\": \"NMR spectroscopy, Ca²⁺ concentration measurements, lipid-binding assays, T cell signaling readouts\",\n      \"pmids\": [\"29058713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether lipid gating operates in all T cell subsets or is tunable by membrane composition unknown\", \"In vivo relevance of the Ca²⁺ gating mechanism not demonstrated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CARMIL2 was established as the specific adaptor linking CD28 to canonical NF-κB activation (but not AP-1 or NFAT), with CARMIL2-deficient patients phenocopying CD28 deficiency features including low memory T cells and recalcitrant warts.\",\n      \"evidence\": \"NF-κB/AP-1/NFAT reporter assays with mutant CARMIL2 alleles, patient immunophenotyping, somatic reversion analysis\",\n      \"pmids\": [\"36515678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of CARMIL2-mediated NF-κB activation downstream of CD28 not fully dissected\", \"Whether CARMIL2 mediates all CD28-dependent NF-κB or a subset unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A previously unappreciated cis-signaling mode was discovered: CD8+ T cells express B7 ligands that engage CD28 in cis at membrane invaginations, driven by PI3K/SNX9-mediated membrane remodeling and signaling through PKCθ to promote survival, migration, and intratumoral persistence.\",\n      \"evidence\": \"Live imaging, PI3K/SNX9 KO and inhibitor studies, PKCθ signaling assays, in vivo mouse tumor models\",\n      \"pmids\": [\"37160118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cis-B7:CD28 signaling occurs in CD4+ T cells or Tregs not tested\", \"Structural basis of cis versus trans B7:CD28 engagement unknown\", \"Quantitative contribution of cis vs trans signaling in tumors not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major open questions include the full-length structure of CD28 in the membrane, the precise mechanism by which CD28 activates chromatin-modifying enzymes at the IL-2 locus, how PI3K exerts both positive metabolic and negative transcriptional effects through the same YMNM motif, and whether the cis-B7:CD28 signaling mode operates broadly across T cell subsets and tissues.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Full-length CD28 structure including transmembrane domain not solved\", \"Chromatin remodeling enzymes recruited by CD28 signaling unidentified\", \"Mechanism of PI3K dual positive/negative roles at YMNM not reconciled\", \"Generality of cis-B7:CD28 signaling across T cell subsets and tissues untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 19, 23]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 14, 25, 27, 29]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 2, 6, 9, 10, 20, 28, 29]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 12, 22, 25, 26]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [12, 24, 26]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD80\",\n      \"CD86\",\n      \"PIK3R1\",\n      \"PIK3R2\",\n      \"GRB2\",\n      \"VAV1\",\n      \"CARMIL2\",\n      \"SNX9\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CD28 is the principal costimulatory receptor on T cells, integrating signals from B7 family ligands to amplify TCR-driven activation, promote survival, and shape metabolic and epigenetic programs that determine effector and memory T cell fate. Identified as a disulfide-bonded homodimeric immunoglobulin superfamily member, CD28 binds B7-1 (CD80), B7-2 (CD86), and—in humans—B7-H2 (ICOSL); upon ligand engagement and tyrosine phosphorylation of its cytoplasmic YMNM motif, it recruits PI3K (p85/p110), Grb2, and adaptors Vav1 and SLP-76 to activate PI3K–Akt signaling, boost PLCγ1/Ca²⁺/NFAT responses, inactivate GSK3, upregulate Bcl-xL for survival, drive glycolysis and early mitochondrial fatty acid oxidation essential for memory formation, and promote epigenetic remodeling of the IL-2 locus [PMID:8146197, PMID:7584133, PMID:12121659, PMID:28919076, PMID:15814687, PMID:8752911]. Transmembrane-level regulation involves acidic phospholipid sequestration of the cytoplasmic domain, relieved by TCR-induced Ca²⁺, creating a dual positive-feedback circuit, while cis-B7:CD28 interactions at invaginated synaptic membranes on CD8⁺ T cells sustain costimulation via PI3K/SNX9/PKCθ to promote intratumoral T cell survival [PMID:29058713, PMID:37160118]. Beyond conventional T cells, CD28 has a cell-intrinsic function in regulatory T cell homeostasis—Treg-specific CD28 deletion causes severe autoimmunity—and transduces pro-survival metabolic signaling in long-lived plasma cells through NF-κB/IRF4-dependent pathways [PMID:23281398, PMID:32579940].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Identification of CD28 (9.3 antigen) as a disulfide-bonded homodimeric T cell surface protein defining a costimulatory activation pathway independent of the TCR/CD3 complex resolved the question of whether T cell activation requires signals beyond the antigen receptor.\",\n      \"evidence\": \"Monoclonal antibody precipitation, co-modulation experiments, and T cell activation assays in primary human T cells\",\n      \"pmids\": [\"3159820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand identity unknown\", \"Signaling mechanism undefined\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 1987,\n      \"claim\": \"Molecular cloning of CD28 cDNA established it as an immunoglobulin superfamily member and confirmed its homodimeric structure, enabling all subsequent structure-function studies.\",\n      \"evidence\": \"COS cell expression cloning with monoclonal antibody binding confirmation\",\n      \"pmids\": [\"2825196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand still unidentified\", \"Cytoplasmic signaling motifs not yet mapped\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Identification of B7 (CD80) as the cognate CD28 ligand with measured binding affinity (~200 nM) established the receptor-ligand pair governing T cell costimulation and IL-2 production.\",\n      \"evidence\": \"Ig fusion protein binding assay, B7-transfected CHO cell costimulation, IL-2 transcript quantification\",\n      \"pmids\": [\"1847722\", \"1650475\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Possibility of additional ligands not excluded\", \"Intracellular signaling pathway downstream of engagement unknown\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Discovery of B7-2 (CD86) as a second CD28/CTLA-4 ligand and generation of CD28-knockout mice collectively demonstrated that CD28 is essential for robust IL-2 production and Ig class switching but that alternative costimulatory pathways exist.\",\n      \"evidence\": \"B7-2 cDNA cloning with CTLA4-Ig binding competition; CD28-KO mice with T cell activation, Ig, and infection phenotyping\",\n      \"pmids\": [\"7694153\", \"7688139\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling intermediates not molecularly defined\", \"Mechanism of alternative costimulation unknown\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Mapping PI3K recruitment to the phosphorylated YMNM motif in the CD28 cytoplasmic tail via p85 SH2 domains—with Y191 mutation abolishing both PI3K binding and IL-2 production—established the central signaling axis of CD28 costimulation.\",\n      \"evidence\": \"Site-directed mutagenesis, baculoviral p85 reconstitution, lipid kinase assay, co-immunoprecipitation in multiple labs\",\n      \"pmids\": [\"8146197\", \"8183372\", \"8080844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PI3K-independent CD28 functions not yet identified\", \"Grb2 contribution not yet dissected from PI3K\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Selective mutagenesis of the YMNM motif separated PI3K and Grb2 binding requirements, showing both are needed for full IL-2 induction and establishing that CD28 nucleates a multi-adaptor signaling complex.\",\n      \"evidence\": \"CD28 point mutants (Y191, M194) with selective PI3K/Grb2 disruption and IL-2 assay using B7-2-transfected cells\",\n      \"pmids\": [\"7584133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream targets of Grb2 in CD28 pathway not defined\", \"Role of additional adaptors (Vav, SLP-76) not yet tested\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstration that CD28 costimulation upregulates Bcl-xL to prevent T cell apoptosis identified the survival arm of CD28 signaling as mechanistically distinct from its proliferative arm.\",\n      \"evidence\": \"CD28-KO T cells with Bcl-xL/Bcl-2 expression analysis, apoptosis assays, ICE protease inhibition\",\n      \"pmids\": [\"8752911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Bcl-xL induction requires PI3K or uses an independent pathway not resolved\", \"Relationship to anergy prevention unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genetic epistasis showed Cbl-b gates CD28 dependence by suppressing Vav activation; Cbl-b loss bypasses the CD28 requirement, placing Vav as a critical downstream node in CD28-dependent T cell activation.\",\n      \"evidence\": \"Cbl-b KO and Cbl-b/CD28 double-KO mice with Vav activation and IL-2 assays\",\n      \"pmids\": [\"10646609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CD28 relieves Cbl-b suppression of Vav not defined\", \"Quantitative contribution of Vav vs. PI3K not separated\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"A Y170F CD28 knockin mouse uncoupled PI3K/Grb2-dependent signals (Bcl-xL, survival) from PI3K-independent functions (anergy prevention, proliferation, IL-2), demonstrating that CD28 engages parallel signaling branches with separable biological outcomes.\",\n      \"evidence\": \"Transgenic knockin mouse expressing CD28-Y170F, T cell activation and anergy assays\",\n      \"pmids\": [\"11276203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the PI3K-independent signaling pathway not molecularly resolved\", \"In vivo infection/autoimmune consequences not fully explored\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"CD28 was shown to amplify TCR signaling specifically at the PLCγ1/Ca²⁺ node (via Itk) without increasing proximal Zap-70 or LAT phosphorylation, defining the point of signal integration between TCR and CD28.\",\n      \"evidence\": \"Biochemical analysis of phospho-Zap70, phospho-LAT, PLCγ1 activation, and Ca²⁺ flux with Itk perturbation\",\n      \"pmids\": [\"11754815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CD28 activates Itk at the molecular level not shown\", \"Whether this applies to all T cell subsets unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Genomic and biochemical studies revealed that CD28 primarily amplifies CD3-initiated transcription rather than activating unique genes, with GSK3 inactivation promoting NFAT nuclear accumulation as a key mechanism, while CD28-PI3K-Akt drives glycolysis and glucose uptake to meet metabolic demands of activation.\",\n      \"evidence\": \"cDNA microarray, GSK3 phosphorylation assay, glucose uptake and glycolysis measurements with PI3K/Akt perturbation\",\n      \"pmids\": [\"12195013\", \"12121659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GSK3 inactivation is PI3K-dependent or PI3K-independent in this context not fully resolved\", \"Metabolic requirements in different T cell subsets not compared\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Biophysical characterization established that CD28 homodimers are monovalent for ligand binding whereas CTLA-4 is bivalent, with B7-2 preferentially binding CD28 over CTLA-4 relative to B7-1, explaining differential costimulatory versus inhibitory receptor engagement.\",\n      \"evidence\": \"Surface plasmon resonance and analytical ultracentrifugation\",\n      \"pmids\": [\"12196291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How monovalent binding translates to signaling threshold not modeled\", \"In vivo kinetic competition between CD28 and CTLA-4 not measured\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Crystal structure of CD28 confirmed the monovalent homodimer architecture and, combined with cryo-EM of mitogenic versus non-mitogenic antibody complexes, constrained models of receptor triggering geometry.\",\n      \"evidence\": \"X-ray crystallography and cryo-electron microscopy\",\n      \"pmids\": [\"15696168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of CD28 bound to B7 ligands available\", \"Transmembrane and cytoplasmic domain conformational changes not captured\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"CD28 costimulation was shown to induce stable epigenetic remodeling of the IL-2 locus (histone acetylation, CpG demethylation, chromatin opening), establishing that CD28 programs the epigenetic state distinguishing activated from anergic T cells.\",\n      \"evidence\": \"Chromatin accessibility, bisulfite sequencing, ChIP for histone marks in activated versus anergic T cells\",\n      \"pmids\": [\"15814687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific chromatin remodelers recruited by CD28 signaling not identified\", \"Whether epigenetic effects are direct or secondary to transcription factor binding unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Discovery that CD28 engagement induces arginine methylation of Vav1, redirecting it to the nucleus, identified a novel post-translational modification linking CD28 to nuclear signaling.\",\n      \"evidence\": \"PRMT activity assay, arginine methylation detection, subcellular fractionation, transmethylation inhibitor\",\n      \"pmids\": [\"16061726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which PRMT is responsible not identified\", \"Nuclear function of methylated Vav1 not characterized\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of B7-H2 (ICOSL) as a third human CD28 ligand using a distinct binding site expanded the ligand repertoire and showed B7-H2:CD28 activates Akt/ERK and upregulates Bcl-xL.\",\n      \"evidence\": \"Receptor array, binding domain mapping, costimulation assays, signaling pathway analysis\",\n      \"pmids\": [\"21530327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Not conserved in mice, limiting genetic model validation\", \"Physiological contexts where B7-H2:CD28 dominates over B7-1/B7-2 not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Treg-specific conditional CD28 deletion revealed a cell-intrinsic post-developmental role for CD28 in maintaining Treg homeostasis and suppressive function, with loss causing severe autoimmunity despite normal Treg numbers.\",\n      \"evidence\": \"Treg-specific CD28 conditional KO mice, flow cytometry, EAE model, complementation experiments\",\n      \"pmids\": [\"23281398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream molecular program by which CD28 maintains Treg suppression not fully mapped\", \"Whether CTLA-4 downregulation is cause or consequence of Treg failure unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"NMR studies revealed that acidic phospholipids sequester the CD28 cytoplasmic domain at the membrane, with TCR-induced Ca²⁺ disrupting this interaction to liberate signaling motifs—establishing a membrane-level gating mechanism for CD28 activation.\",\n      \"evidence\": \"NMR spectroscopy of CD28 TM/cytoplasmic domain, lipid binding assays, Ca²⁺ perturbation, T cell signaling readouts\",\n      \"pmids\": [\"29058713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether lipid composition variations across T cell subsets modulate this gating is unknown\", \"In vivo confirmation of the dual feedback circuit not yet achieved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"CD28 was shown to transiently induce CPT1a before first division, priming mitochondrial fatty acid oxidation and spare respiratory capacity essential for memory T cell formation, with miR-33 attenuating this program in the absence of CD28.\",\n      \"evidence\": \"CD28-deficient T cells, CPT1a expression kinetics, miR-33/TXNIP analysis, mitochondrial imaging, spare respiratory capacity measurement\",\n      \"pmids\": [\"28919076\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling branch (PI3K vs. PI3K-independent) driving CPT1a induction not identified\", \"Whether this metabolic priming applies to CD4⁺ T cells equally is unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"CD28 was found to transduce pro-survival signals in long-lived plasma cells through SLP-76-dependent NF-κB activation driving IRF4 and a ROS-dependent metabolic program, extending CD28 function beyond T cells.\",\n      \"evidence\": \"LLPC vs. SLPC comparison, SLP76 expression, glucose uptake, mitochondrial/ROS assays, NF-κB and IRF4 measurement\",\n      \"pmids\": [\"32579940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CD28 ligand source in the bone marrow niche not defined\", \"Whether plasma cell CD28 signaling uses the same YMNM-PI3K axis as T cells not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery of cis-B7:CD28 interactions on CD8⁺ T cells at invaginated synaptic membranes—driven by PI3K and SNX9 and signaling through PKCθ—revealed a cell-autonomous costimulatory mechanism that sustains intratumoral T cell survival.\",\n      \"evidence\": \"Live cell imaging, co-IP, PI3K/SNX9 perturbation, PKCθ assay, T cell-specific B7 KO in mouse tumor models\",\n      \"pmids\": [\"37160118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of cis vs. trans B7:CD28 interaction not resolved\", \"Whether cis-signaling occurs in CD4⁺ T cells or Tregs unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of the CD28–B7 ligand complex, the molecular identity of the PI3K-independent CD28 signaling branch that prevents anergy, the chromatin remodelers directly recruited by CD28 signaling, and how cis- versus trans-B7 engagement is regulated across immune cell types and tissue microenvironments.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length CD28:B7 co-crystal structure exists\", \"PI3K-independent anergy-prevention pathway molecularly uncharacterized\", \"Tissue-specific regulation of cis-B7:CD28 not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [1, 2, 3, 5, 7, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7, 10, 39]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 21, 31, 34, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 3, 5, 7, 8, 10, 11, 12, 13, 15, 26, 28, 35]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 8, 10, 17, 26, 27, 31, 36]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [17, 32, 42]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [11, 36]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD80\",\n      \"CD86\",\n      \"PIK3R1\",\n      \"GRB2\",\n      \"VAV1\",\n      \"SLP76\",\n      \"PRKCQ\",\n      \"ITK\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}