{"gene":"CD6","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1991,"finding":"CD6 is a type I integral membrane glycoprotein with an extracellular domain containing scavenger receptor cysteine-rich (SRCR) domains, and its cytoplasmic domain contains serine residues that are substrates for phosphorylation during T cell activation.","method":"cDNA cloning, COS cell transfection, RNA blot hybridization, phosphorylation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — full-length cDNA isolation with functional expression and biochemical characterization","pmids":["1919444"],"is_preprint":false},{"year":1989,"finding":"CD6 (T12) is a 130 kDa glycoprotein on T cells that, when cross-linked with anti-CD3, delivers co-activating signals to CD4+ T cells via an IL-2-dependent pathway, and its activation is macrophage-dependent.","method":"In vitro T cell proliferation assays, IL-2R expression measurement, anti-IL-2R inhibition, anti-CD3 cross-linking","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple functional readouts with defined cellular phenotype, replicated by multiple labs","pmids":["2794503"],"is_preprint":false},{"year":1989,"finding":"CD6 is a monomeric 130 kDa glycoprotein under reducing conditions with intrachain disulfide bonds, is extensively N-glycosylated, and is serine-phosphorylated in activated T cells; two anti-CD6 mAbs recognizing distinct epitopes deliver different activation signals linked to different cofactor requirements (macrophages vs. PMA).","method":"Surface radiolabeling, immunoprecipitation, Western blot, biosynthetic labeling with tunicamycin, phosphorylation assays","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 1 — multiple biochemical methods characterizing structure and post-translational modifications","pmids":["2481822"],"is_preprint":false},{"year":1990,"finding":"CD6 surface expression is regulated by phosphorylation: PKC activation rapidly converts an unphosphorylated 105 kDa form to a phosphorylated 130 kDa form without requiring new protein synthesis; this interconversion is reversible and alkaline phosphatase treatment converts the 130 kDa form back to 105 kDa.","method":"Surface 125I labeling, immunoprecipitation, 32P labeling, alkaline phosphatase treatment, PKC activator stimulation","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical demonstration of reversible phosphorylation-dependent isoform conversion","pmids":["2384666"],"is_preprint":false},{"year":1991,"finding":"CD6 biosynthesis proceeds through an 88 kDa nascent polypeptide, an immature N-glycosylated 110 kDa intermediate, and a mature 130 kDa surface form bearing sulfated O-linked oligosaccharides; CD6 is phosphorylated in resting cells and can be hyperphosphorylated by PKC activators.","method":"Pulse-chase biosynthetic labeling, tunicamycin treatment, 32P labeling, immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — detailed biosynthetic characterization with multiple orthogonal biochemical methods","pmids":["2016320"],"is_preprint":false},{"year":1993,"finding":"CD6 cytoplasmic tyrosine residues become phosphorylated upon TCR/CD3 stimulation; co-crosslinking with CD4 produces the highest level of CD6 tyrosine phosphorylation, while co-crosslinking with CD2 also augments it, but crosslinking CD2, CD4, or CD28 alone does not phosphorylate CD6.","method":"Anti-phosphotyrosine immunoprecipitation, co-crosslinking of CD3 with CD2, CD4, or CD28 on primary T cells","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical demonstration of TCR-dependent tyrosine phosphorylation with defined cofactor requirements","pmids":["7678115"],"is_preprint":false},{"year":1995,"finding":"CD6 has a large cytoplasmic domain of 244 amino acids (previously underestimated) containing two proline-rich SH3-binding motifs, serine-threonine-rich repeats, PKC phosphorylation sites, and casein kinase-2 sites; alternatively spliced cytoplasmic isoforms exist in human peripheral blood lymphocytes.","method":"RT-PCR, cDNA cloning and sequencing, COS cell transfection, immunoprecipitation, SDS-PAGE","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — sequence-based domain identification validated by functional expression","pmids":["7589069"],"is_preprint":false},{"year":1995,"finding":"ALCAM (CD166) is a CD6 ligand: COS cells expressing CD6 adhere to thymic epithelial cells, this adhesion is blocked by anti-CD6 mAb, and an ALCAM-Rg fusion protein binds specifically to CD6 transfectants; ALCAM-CD6 interaction mediates thymocyte–thymic epithelial cell adhesion.","method":"COS cell transfection, cell adhesion assays, antibody blocking, immunoglobulin fusion protein binding, cDNA cloning","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — reconstituted receptor-ligand interaction with reciprocal binding and functional adhesion assay","pmids":["7760007"],"is_preprint":false},{"year":1995,"finding":"The membrane-proximal SRCR domain (domain 3) of CD6 contains the ALCAM binding site; mAbs binding to this domain preferentially block CD6-ALCAM binding.","method":"Domain-specific CD6-Rg fusion proteins, cell adhesion assays, antibody blocking experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — systematic domain-deletion fusion protein analysis with functional adhesion readout","pmids":["7543097"],"is_preprint":false},{"year":1996,"finding":"The amino-terminal Ig-like domain of ALCAM specifically binds the membrane-proximal (third) SRCR domain of CD6 in a 1:1 stoichiometry, representing the first characterized Ig-domain/SRCR-domain protein interaction.","method":"Truncated Ig fusion proteins, receptor-ligand binding assays, thrombin cleavage to produce monomeric domains, stoichiometry determination","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted interaction with defined stoichiometry using truncated recombinant proteins","pmids":["8663238"],"is_preprint":false},{"year":1997,"finding":"Three residues in the membrane-proximal SRCR domain (domain 3) of CD6, located in a region of low sequence conservation, when mutated abolish ALCAM binding without disrupting overall CD6 conformation (as shown by intact mAb binding); these are the first residues critical for SRCR domain-ligand interaction.","method":"Site-directed mutagenesis, binding assays with anti-CD6 mAbs, ALCAM binding assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with orthogonal structural controls identifies specific binding residues","pmids":["9054570"],"is_preprint":false},{"year":1996,"finding":"Residues critical for CD6 binding in ALCAM cluster on the predicted A'GFCC'C\" face of ALCAM's N-terminal Ig domain, identified by targeted mutagenesis; this site is conserved across species.","method":"Targeted mutagenesis of ALCAM, CD6-ALCAM binding assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis-based mapping of binding site with structural modeling","pmids":["8823162"],"is_preprint":false},{"year":2004,"finding":"CD6 interacts with CD166 (ALCAM) with a KD of 0.4–1.0 μM and fast off-rate; soluble monomeric CD6 or CD166 at concentrations blocking this interaction inhibit antigen-specific human T cell responses, demonstrating that extracellular CD6-CD166 engagement is required for optimal immune response.","method":"Surface plasmon resonance (SPR) kinetics, soluble protein inhibition of antigen-specific T cell proliferation assays","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — quantitative binding kinetics plus functional inhibition with defined concentrations","pmids":["15048703"],"is_preprint":false},{"year":1997,"finding":"CD6 gene is located on chromosome 11q13 near CD5 and CD20; it is encoded by at least 13 exons with each SRCR domain encoded by a separate exon; at least five cytoplasmic domain isoforms exist from alternative splicing of cytoplasmic exons.","method":"Genomic cloning, FISH, YAC library screening, RT-PCR, mRNA analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — genomic characterization with direct structural evidence for splice isoforms","pmids":["9013954"],"is_preprint":false},{"year":1997,"finding":"CD6 cytoplasmic tyrosine residues Y629 and Y662 (the two C-terminal tyrosines) are critical for TCR-induced tyrosine phosphorylation; isoform CD6e lacking proline-rich motifs is not phosphorylated; Ca2+ mobilization by CD6/TCR co-ligation requires a membrane-proximal cytoplasmic region N-terminal to amino acid 555, distinct from the tyrosine phosphorylation region.","method":"Chimeric receptor expression, TCR cross-linking, anti-phosphotyrosine immunoprecipitation, Ca2+ flux measurement, site-directed mutagenesis","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis of specific residues with functional phosphorylation and calcium flux readouts","pmids":["9394826"],"is_preprint":false},{"year":2004,"finding":"CD6 physically associates with the TCR/CD3 complex (shown by co-immunoprecipitation, co-capping, and FRET); CD6 and ALCAM colocalize with TCR/CD3 at the central SMAC of the immunological synapse; soluble CD6 reduces T cell-APC conjugate formation and inhibits CD3-mediated T cell proliferation in a dose-dependent manner.","method":"Co-immunoprecipitation, co-capping, FRET, confocal microscopy of Ag-specific conjugates, proliferation inhibition assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods demonstrating physical association and immunological synapse localization with functional consequence","pmids":["15294938"],"is_preprint":false},{"year":2002,"finding":"CD5 and CD6 physically associate at the lymphocyte cell membrane, co-immunoprecipitate from Brij 96 detergent lysates independently of other surface receptors, and co-accumulate at the immunological synapse; this association is independent of the CD5 cytoplasmic region.","method":"Co-immunoprecipitation, FRET, co-capping, co-modulation experiments, imaging of T cell-APC conjugates","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus FRET and co-capping demonstrating physical in vivo association","pmids":["12473675"],"is_preprint":false},{"year":2003,"finding":"CD6 (rat OX52) co-precipitates with CD5, and the CD5 fraction associated with CD6 is highly phosphorylated compared to CD5 associated with TCR or CD2; CD6 uniquely associates with Lck, Fyn, ZAP-70, and the Tec-family kinase Itk (not found in CD2/CD5/TCR complexes); Lck+Itk effectively phosphorylate CD5 cytoplasmic peptides.","method":"Immunoprecipitation, in vitro kinase assays with synthetic peptides, RT-PCR cloning of rat CD6","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP with kinase assays identifying unique Itk association and CD5 phosphorylation role","pmids":["12525577"],"is_preprint":false},{"year":2005,"finding":"CD6 and ALCAM are actively recruited to the DC-T cell contact zone; ALCAM-blocking antibodies interfere with DC-T cell conjugate formation; CD6-ALCAM interactions are required during the proliferative phase of T cell response; CD6 cross-linking with CD3 enhances proliferation to levels similar to CD28 co-stimulation.","method":"Fluorescence microscopy of DC-T cell contacts, antibody blocking of conjugate formation, T cell proliferation assays with blocking antibodies and recombinant ALCAM-Fc, CD6/CD3 co-crosslinking","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple functional assays demonstrating adhesion and costimulatory roles with defined mechanistic interventions","pmids":["16352806"],"is_preprint":false},{"year":2005,"finding":"The cytoplasmic tail of CD6 interacts with syntenin-1, a PDZ domain-containing scaffolding protein, through CD6's C-terminal sequence (-YDDISAA) and both PDZ domains of syntenin-1; syntenin-1 accumulates at CD6 caps and at the immunological synapse.","method":"Yeast two-hybrid screen, mutational analysis, pull-down assays, co-immunoprecipitation in mammalian cells, confocal microscopy","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid discovery confirmed by pull-down, co-IP, and mutagenesis in mammalian cells","pmids":["16034076"],"is_preprint":false},{"year":2006,"finding":"CD6 ligation recruits the adaptor SLP-76 through phosphorylation-dependent binding to tyrosine 662 (Y662) in the CD6 cytoplasmic domain (KD = 0.5 μM at 37°C); this interaction mediates CD6 costimulation; Y662F mutation abolishes both SLP-76 binding and costimulation; CD6 and SLP-76 co-precipitate from normal human T cells.","method":"Phosphopeptide binding assay (SPR/equilibrium), co-precipitation from primary T cells, CD6 cytoplasmic mutant analysis in T cell hybridoma model","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — direct binding kinetics of phosphopeptide to SLP-76 plus mutagenesis and co-IP in primary cells","pmids":["16914752"],"is_preprint":false},{"year":2006,"finding":"CD6 ligation (by mAbs or ALCAM-Fc) induces time- and dose-dependent activation of ERK1/2, p38, and JNK MAPK cascades in T cells; this requires the C-terminal cytoplasmic region of CD6 and Src tyrosine kinases; CD6 ligation activates AP-1 and c-Fos SRE transcription; synergistic effects occur with TCR/CD3 co-ligation.","method":"Western blot for phospho-MAPK, reporter gene assays (SRE-luciferase, AP-1-luciferase), Src kinase inhibitors, mAb and ALCAM-Fc stimulation","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple MAPK pathways confirmed with pharmacological inhibitors and reporter assays","pmids":["16818773"],"is_preprint":false},{"year":2007,"finding":"A CD6 isoform lacking SRCR domain 3 (CD6Δd3), generated by exon 5 skipping, cannot localize to the immunological synapse (unlike full-length CD6), demonstrating that SRCR domain 3 (the ALCAM-binding domain) is required for CD6 targeting to the T cell-APC interface; CD6Δd3 is upregulated upon T cell activation.","method":"cDNA cloning, flow cytometry, confocal imaging of immunological synapse, RT-PCR at single-cell level, Western blot","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — direct imaging of IS localization with domain deletion mutant and multiple expression analyses","pmids":["17371992"],"is_preprint":false},{"year":2007,"finding":"CD6 binds lipoteichoic acid (Gram-positive bacteria) and LPS (Gram-negative bacteria) through its ectodomain; the KD for LPS-CD6 interaction is ~2.69×10⁻⁸ M; membrane CD6 also retains LPS-binding ability and activates the MAPK signaling cascade upon LPS binding; recombinant soluble CD6 protects mice from lethal LPS challenge and reduces TNF-α, IL-6, and IL-1β.","method":"Binding assays with recombinant soluble CD6, bacterial aggregation, SPR/affinity measurement, MAPK activation assays, in vivo LPS challenge mouse model, serum cytokine measurement","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding with quantified affinity plus in vivo functional protection, multiple methods","pmids":["17601777"],"is_preprint":false},{"year":2011,"finding":"CD6 significantly attenuates early and late TCR signaling: expression of CD6 (but not a cytoplasmic domain deletion mutant) reduces calcium mobilization and IL-2 release after superantigen or anti-CD3 stimulation; morpholino-mediated knockdown of CD6 enhances calcium signals; blocking CD6-CD166 interaction with anti-CD166 increases T cell proliferation, but anti-CD6 antibodies inhibit it.","method":"Single-cell calcium imaging, IL-2 ELISA, Jurkat overexpression of CD6 vs. cytoplasmic deletion mutant, morpholino knockdown in primary human T cells, antibody blocking proliferation assays","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple complementary approaches (overexpression, KD, deletion mutant) with early and late signaling readouts","pmids":["21956609"],"is_preprint":false},{"year":2014,"finding":"Quantitative mass spectrometry of primary mouse T cells revealed that CD6 recruits SLP-76 and Vav1 independently of the LAT adaptor, constituting a LAT-independent TCR signaling hub; 112 high-confidence time-resolved protein interactions around Zap70, Lat, and SLP-76 were mapped.","method":"Knock-in affinity tag mass spectrometry (quantitative proteomics) in primary CD4+ T cells, time-resolved interaction mapping","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1 — knock-in quantitative MS with time resolution in primary cells, identifies LAT-independent CD6-SLP-76-Vav1 pathway","pmids":["24584089"],"is_preprint":false},{"year":2015,"finding":"X-ray crystal structures of CD6 (three SRCR domains) and CD166 (two N-terminal domains) reveal a nonlinear organization of consecutive SRCR domains; a disease-associated SNP in CD6 introduces a glycosylation site that sterically hinders the CD6/CD166 interaction; native MS shows competition between heterophilic CD6-CD166 and homophilic CD166-CD166 interactions.","method":"X-ray crystallography, native mass spectrometry, glycosylation site mutagenesis, binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus native MS and mutagenesis providing mechanistic insight into SNP effect","pmids":["26146185"],"is_preprint":false},{"year":2014,"finding":"ALCAM recruitment to adhesion sites and its membrane tether-anchoring propensity depend on actin cytoskeletal interactions; linking ALCAM to the actin cortex strengthens CD6-mediated cell adhesion and stiffens the cortex, forming a mechanical link between CD6 (on T cells) and the actin cortex (via ALCAM on DCs) at the immunological synapse.","method":"Single-cell force spectroscopy, TIRF microscopy, ALCAM cytoplasmic tail mutation constructs, actin cortex stiffness measurements","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 — reconstituted adhesion mechanics with cytoplasmic domain mutants and biophysical measurements","pmids":["24496453"],"is_preprint":false},{"year":2014,"finding":"T cell activation regulates CD6 alternative splicing (exon 5 skipping to produce CD6Δd3) through increased RNA Pol II occupancy and chromatin acetylation; the splicing factor SRSF1 binds CD6 intron 4 to promote exon 5 inclusion, and upon activation SRSF1 is downregulated and its recruitment to CD6 transcript is impaired by increased chromatin acetylation.","method":"Chromatin immunoprecipitation (ChIP) for RNA Pol II and acetylation, RNA immunoprecipitation (RIP) for SRSF1, histone deacetylase inhibitor experiments, RT-PCR","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — ChIP and RIP with mechanistic intervention (HDAC inhibitor) identifying SRSF1 as splicing regulator","pmids":["24890719"],"is_preprint":false},{"year":2017,"finding":"CD6 cytoplasmic Y629 residue recruits the SH2 adaptor GADS; bivalent recruitment of a GADS/SLP-76 complex (at Y629 and Y662 respectively) is required for CD6 costimulation; both Y629F and Y662F mutations abolish costimulation in Jurkat and primary T cells.","method":"Biochemical pulldown, mutational analysis of Y629F and Y662F in Jurkat and primary T cells, T cell activation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with functional costimulation readout identifying bivalent GADS/SLP-76 recruitment mechanism","pmids":["28289074"],"is_preprint":false},{"year":2017,"finding":"CD318 is a CD6 ligand distinct from CD166: identified using mAb 3A11 immunoprecipitation and proteomics; CD318 KO mice are protected from experimental autoimmune encephalomyelitis (like CD6 KO mice); CD318 mediates CD6-dependent adhesion of T cells to synovial fibroblasts.","method":"Mass spectrometry protein identification, CD318 KO mouse EAE model, T cell adhesion assays to synovial fibroblasts, soluble CD318 chemotaxis assay","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — MS identification of ligand confirmed by KO phenotype and functional adhesion assays","pmids":["28760953"],"is_preprint":false},{"year":2021,"finding":"CRISPR/Cas9-based quantitative MS of primary mouse T cells defines the CD6 signalosome as comprising both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation; CD6 also associates constitutively (independently of TCR engagement) with proteins supporting T cell transendothelial migration.","method":"CRISPR/Cas9 knock-in in primary mouse T cells, quantitative mass spectrometry, TCR stimulation time-course","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 — primary cell CRISPR knock-in with quantitative MS identifying dual positive/negative signalosome composition","pmids":["33125054"],"is_preprint":false},{"year":2016,"finding":"CD6 deficiency in mice reduces CD4+ and CD8+ single-positive thymocyte numbers; CD6−/− double-positive thymocytes show increased Ca2+ mobilization to TCR cross-linking; bone marrow chimera experiments reveal a T cell-autonomous selective disadvantage of CD6−/− cells during development; CD6−/− mice show increased effector/memory and regulatory T cells but diminished Treg suppressive activity.","method":"CD6 KO mouse analysis, bone marrow chimeras, TCR-transgenic crosses, Ca2+ flux assays, Treg suppression assays, collagen-induced arthritis model","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — comprehensive in vivo KO analysis with bone marrow chimeras and multiple T cell subset phenotyping","pmids":["27377588"],"is_preprint":false},{"year":2017,"finding":"CD6 KO mice show decreased pathogenic T cell responses, reduced spinal cord T cell infiltration, and attenuated EAE; CD6-deficient T cells show augmented initial activation but reduced survival and proliferation, leading to decreased Th1 and Th17 polarization; activated CD6-deficient T cells show impaired infiltration through brain microvascular endothelial cell monolayers.","method":"CD6 KO mouse EAE model, T cell recall assays, Th1/Th17 polarization assays, transendothelial migration assay, CD6-humanized mouse treatment with anti-human CD6 mAb","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with multiple defined functional phenotypes plus humanized mouse treatment validation","pmids":["28209777"],"is_preprint":false},{"year":1997,"finding":"CD6 ligation protects chronic lymphocytic leukemia B cells from anti-IgM-induced apoptosis by downregulating bax-α mRNA levels and preventing bcl-2 downregulation, resulting in an increased Bcl-2/Bax ratio.","method":"Anti-CD6 crosslinking on B-CLL cells, Northern blot for bax-α and bcl-2 mRNA, apoptosis assays","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 — direct mechanistic link from CD6 ligation to apoptosis resistance via Bcl-2/Bax ratio, single lab","pmids":["9108402"],"is_preprint":false},{"year":2014,"finding":"CD6 and CD166/ALCAM interact with Galectin-1 and Galectin-3 in a carbohydrate-dependent manner; galectins interfere with superantigen-induced T cell proliferation and CD6-CD166-mediated cell adhesion; CD6 expression protects cells from galectin-induced apoptosis.","method":"Galectin binding assays, T cell proliferation assays with galectin addition, cell adhesion assays, apoptosis measurement","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 — binding and functional assays in single study without detailed mechanistic dissection","pmids":["24945728"],"is_preprint":false},{"year":2016,"finding":"CD6 is selectively expressed on B1 cells outside the bone marrow and peritoneal cavity; CD6−/− mice are protected from intestinal ischemia/reperfusion injury, with reduced natural IgM titers and B1a cell populations, demonstrating a role for CD6 in B1a cell self-renewal.","method":"CD6 KO mouse intestinal I/R model, flow cytometry for B1a cells, serum natural IgM ELISA","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with mechanistic link to B1a self-renewal and IgM production, single study","pmids":["27909060"],"is_preprint":false},{"year":1994,"finding":"PKC activation by phorbol ester (PMA) increases CD6 surface expression by increasing CD6 mRNA transcription (nuclear run-on); this requires new protein synthesis; CD2 ligation (but not ligation of other surface molecules tested) upregulates CD6 expression on thymocytes but not mature T cells.","method":"Northern blot, nuclear run-on transcription assays, Western blot, flow cytometry, PKC inhibitor studies, actinomycin D and cycloheximide experiments","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — nuclear run-on directly measures transcription rate; multiple controls including translation inhibitors","pmids":["8207228"],"is_preprint":false},{"year":2004,"finding":"A second CD6 ligand (3A11 antigen, later identified as CD318) distinct from CD166 is expressed on cells from thymus, skin, synovium, and cartilage; its expression is enhanced by IFN-γ; it has a molecular mass of 130 kDa and is immunoprecipitated by soluble CD6-Ig fusion protein; anti-CD166 siRNA knockdown does not alter 3A11 expression.","method":"Immunoprecipitation with CD6-Ig fusion protein, confocal microscopy, siRNA knockdown of CD166, flow cytometry, T cell adhesion assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — distinct ligand confirmed by reciprocal immunoprecipitation and siRNA exclusion of CD166, single lab","pmids":["15528349"],"is_preprint":false},{"year":2017,"finding":"Itolizumab (anti-CD6 D1 mAb) directly inhibits CD6 receptor hyperphosphorylation and decreases associated ZAP70 kinase and SLP76 docking protein levels; F(ab')2 fragment of Itolizumab does not produce this inhibition, indicating the intact antibody format is required for the mechanism.","method":"CD6 phosphorylation Western blot, ZAP70 and SLP76 co-immunoprecipitation, Itolizumab vs. F(ab')2 comparison","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical measurement of phosphorylation and signaling complex, single lab","pmids":["28672038"],"is_preprint":false}],"current_model":"CD6 is a type I transmembrane SRCR superfamily glycoprotein expressed primarily on T cells that functions as a multitask signaling hub at the immunological synapse: its membrane-proximal SRCR domain 3 binds ligands CD166/ALCAM and CD318 (and pathogen-associated patterns including LPS), recruiting CD6 to the TCR/CD3 complex at the central SMAC; TCR activation induces tyrosine phosphorylation of cytoplasmic residues Y629 and Y662, enabling bivalent recruitment of a GADS/SLP-76 complex (costimulatory) and independently of LAT recruits Vav1, while also associating with the negative regulator UBASH3A/STS-2, making CD6 both a positive and negative rheostat of T cell activation; alternative splicing of exon 5 (regulated by SRSF1 and chromatin acetylation upon activation) produces a CD6Δd3 isoform that lacks the ALCAM-binding domain and cannot localize to the immunological synapse, providing a mechanism to tune CD6-mediated signaling."},"narrative":{"teleology":[{"year":1989,"claim":"Establishing CD6 as a T cell costimulatory molecule: prior to this, CD6 (T12) was an orphan 130 kDa T cell surface glycoprotein; cross-linking with anti-CD3 demonstrated that it delivers IL-2-dependent co-activating signals to CD4+ T cells, requiring macrophage cofactors.","evidence":"In vitro T cell proliferation and IL-2R expression assays with anti-CD6/anti-CD3 co-crosslinking","pmids":["2794503","2481822"],"confidence":"High","gaps":["Costimulatory ligand unknown","Signaling pathway downstream of CD6 uncharacterized","Contribution of macrophages mechanistically undefined"]},{"year":1991,"claim":"Molecular cloning and biochemical characterization revealed that CD6 is an SRCR superfamily member with extracellular SRCR domains and a cytoplasmic domain subject to serine phosphorylation and PKC-regulated electrophoretic mobility shifts, establishing its signaling potential.","evidence":"cDNA cloning, COS cell transfection, pulse-chase biosynthetic labeling, reversible phosphatase treatment","pmids":["1919444","2384666","2016320"],"confidence":"High","gaps":["Tyrosine phosphorylation not yet examined","Ligand not identified","Relationship to TCR signaling unknown"]},{"year":1995,"claim":"Identification of ALCAM/CD166 as the CD6 ligand and mapping the interaction to SRCR domain 3 resolved the long-standing question of what CD6 binds, establishing this as the first characterized SRCR–Ig-domain receptor–ligand pair.","evidence":"COS cell adhesion assays, Ig fusion protein binding, domain-deletion constructs, mAb blocking","pmids":["7760007","7543097","8663238"],"confidence":"High","gaps":["Binding affinity not yet quantified","In vivo relevance of CD6-ALCAM interaction unproven","Additional ligands suspected but not identified"]},{"year":1997,"claim":"Identification of Y629 and Y662 as the TCR-induced phosphorylation sites in the CD6 cytoplasmic domain, and demonstration that calcium mobilization maps to a distinct membrane-proximal region, established that CD6 signaling involves separable functional modules.","evidence":"Site-directed mutagenesis of chimeric receptors, anti-phosphotyrosine IP, calcium flux measurement in T cell lines","pmids":["9394826","7678115","9013954"],"confidence":"High","gaps":["Downstream adaptor proteins binding phosphotyrosines unknown","In vivo significance of individual tyrosines untested","Contribution of alternatively spliced cytoplasmic isoforms unclear"]},{"year":2004,"claim":"Quantitative kinetic measurement of CD6–ALCAM interaction (KD 0.4–1.0 µM, fast off-rate) and demonstration that CD6 physically associates with TCR/CD3 at the central SMAC placed CD6 as an integral component of the immunological synapse required for optimal T cell responses.","evidence":"Surface plasmon resonance, co-immunoprecipitation, FRET, confocal microscopy of antigen-specific conjugates, soluble protein inhibition of proliferation","pmids":["15048703","15294938"],"confidence":"High","gaps":["Mechanism of CD6 recruitment to TCR complex unknown","Role of second ligand (3A11/CD318) at IS undefined","Structural basis of CD6-TCR association unclear"]},{"year":2006,"claim":"Discovery that phospho-Y662 recruits SLP-76 directly and that CD6 ligation activates ERK1/2, p38, JNK, and AP-1 pathways identified the core costimulatory signaling cascade downstream of CD6.","evidence":"Phosphopeptide–SLP-76 SPR binding, Y662F mutagenesis abolishing costimulation, phospho-MAPK Western blots, reporter gene assays with Src kinase inhibitors","pmids":["16914752","16818773"],"confidence":"High","gaps":["Whether SLP-76 recruitment to CD6 is LAT-dependent or independent not resolved","Identity of adaptor binding Y629 unknown","MAPK activation mechanism via CD6 vs. TCR not dissected"]},{"year":2007,"claim":"Discovery of the CD6Δd3 splice isoform (lacking exon 5/SRCR domain 3) that cannot localize to the immunological synapse and is upregulated upon T cell activation established a built-in feedback mechanism to modulate CD6-mediated costimulation.","evidence":"RT-PCR, confocal imaging of IS localization with domain-deleted construct, single-cell splice variant analysis","pmids":["17371992"],"confidence":"High","gaps":["Splicing regulation mechanism unknown","Functional consequence of CD6Δd3 upregulation on T cell effector function untested","Whether CD6Δd3 acts as dominant negative unclear"]},{"year":2007,"claim":"Demonstration that CD6 ectodomain binds bacterial LPS and lipoteichoic acid with nanomolar affinity and that soluble CD6 protects mice from lethal endotoxemia revealed an innate immune pattern-recognition function for CD6 beyond adaptive T cell costimulation.","evidence":"Recombinant soluble CD6 binding assays, SPR affinity measurement (KD ~27 nM for LPS), in vivo LPS challenge with survival and cytokine readouts","pmids":["17601777"],"confidence":"High","gaps":["Which SRCR domain binds LPS not mapped","Signaling pathway triggered by LPS–CD6 on T cells not dissected separately from TCR signals","Physiological relevance of CD6 pathogen sensing in infection models untested"]},{"year":2014,"claim":"Quantitative proteomics in primary T cells revealed that CD6 recruits SLP-76 and Vav1 independently of the canonical adaptor LAT, establishing CD6 as a LAT-independent TCR signaling node, while concurrent work identified SRSF1 and chromatin acetylation as regulators of CD6 exon 5 alternative splicing.","evidence":"Knock-in affinity-tag MS in primary mouse CD4+ T cells; ChIP for Pol II/acetylation and RIP for SRSF1 with HDAC inhibitor perturbation","pmids":["24584089","24890719"],"confidence":"High","gaps":["How GADS fits into the LAT-independent pathway not yet shown","Whether Vav1 recruitment is direct or SLP-76-mediated unclear","Epigenetic regulation of CD6 splicing in disease states untested"]},{"year":2016,"claim":"CD6 knockout mouse analysis revealed that CD6 deficiency causes augmented initial TCR calcium signaling but reduced thymocyte numbers and impaired Treg function, establishing CD6 as a dual positive/negative modulator of T cell development and activation in vivo.","evidence":"CD6 KO mice with bone marrow chimeras, TCR-transgenic crosses, calcium flux, Treg suppression assays, collagen-induced arthritis model","pmids":["27377588"],"confidence":"High","gaps":["Molecular basis of enhanced calcium signaling in CD6-deficient cells undefined","Mechanism of impaired Treg function unclear","Whether phenotype is ALCAM-dependent or CD318-dependent not addressed"]},{"year":2017,"claim":"Identification of CD318 as a second CD6 ligand, demonstration that bivalent GADS(Y629)/SLP-76(Y662) recruitment is required for costimulation, and CD6 KO protection from EAE with impaired T cell transendothelial migration collectively established CD6 as a multi-ligand receptor integrating costimulation with tissue infiltration.","evidence":"MS-based CD318 identification, CD318 KO EAE model, Y629F/Y662F mutagenesis in Jurkat and primary T cells, brain endothelial transmigration assays","pmids":["28760953","28289074","28209777"],"confidence":"High","gaps":["Which CD6 domain binds CD318 not mapped","Relative contributions of ALCAM vs. CD318 to IS formation undefined","Transendothelial migration mechanism downstream of CD6 not molecularly dissected"]},{"year":2021,"claim":"CRISPR knock-in quantitative MS defined the full CD6 signalosome as containing both positive regulators (SLP-76, ZAP70, VAV1) and the negative regulator UBASH3A/STS-2, plus constitutive associations supporting transendothelial migration, resolving the paradox of CD6 serving as both costimulatory and inhibitory.","evidence":"CRISPR/Cas9 knock-in in primary mouse T cells with time-resolved quantitative MS after TCR stimulation","pmids":["33125054"],"confidence":"High","gaps":["Mechanism by which UBASH3A counteracts CD6 costimulation unknown","Stoichiometry of positive vs. negative regulator recruitment not determined","Whether constitutive CD6 associations change during inflammation not tested"]},{"year":null,"claim":"Key unresolved questions include: how UBASH3A-mediated negative regulation mechanistically opposes GADS/SLP-76 costimulation; which SRCR domain(s) bind CD318; how CD6 dual signaling is partitioned during thymic selection versus peripheral activation; and whether CD6Δd3 actively suppresses signaling or merely fails to engage the synapse.","evidence":"","pmids":[],"confidence":"Low","gaps":["UBASH3A inhibitory mechanism via CD6 uncharacterized","CD318 binding domain on CD6 unmapped","No structural model of full-length CD6 cytoplasmic domain with adaptors","Functional role of CD6Δd3 beyond failure to localize is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[20,29,31]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[24,32,33]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[7,12,18,27]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,7,15,22]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,18,24,31,32,33]}],"complexes":["TCR/CD3 complex (recruited upon activation)","CD5-CD6 complex"],"partners":["ALCAM","CDCP1","SLP76","GADS","VAV1","UBASH3A","CD5","SDCBP"],"other_free_text":[]},"mechanistic_narrative":"CD6 is a type I transmembrane glycoprotein of the scavenger receptor cysteine-rich (SRCR) superfamily that functions as a dual costimulatory and inhibitory signaling hub at the T cell immunological synapse, integrating adhesion, pathogen sensing, and TCR signal modulation. Its membrane-proximal SRCR domain 3 binds the ligands CD166/ALCAM (KD ~0.4–1.0 µM) and CD318, mediating T cell–APC adhesion and targeting CD6 to the central SMAC where it physically associates with the TCR/CD3 complex and CD5 [PMID:7760007, PMID:15294938, PMID:28760953, PMID:12473675]. TCR engagement triggers tyrosine phosphorylation of cytoplasmic residues Y629 and Y662, which bivalently recruit a GADS/SLP-76 signalosome independently of LAT, while also engaging the negative regulator UBASH3A/STS-2, thereby enabling CD6 to both promote and attenuate T cell activation depending on context; CD6 deficiency in mice causes augmented initial TCR signaling but impaired T cell survival, reduced Th1/Th17 responses, and protection from EAE [PMID:28289074, PMID:24584089, PMID:33125054, PMID:27377588, PMID:28209777]. T cell activation induces alternative splicing of exon 5 (regulated by SRSF1 and chromatin acetylation) to produce a CD6Δd3 isoform lacking the ALCAM-binding domain that cannot localize to the immunological synapse, providing a feedback mechanism to tune CD6-mediated costimulation [PMID:17371992, PMID:24890719]."},"prefetch_data":{"uniprot":{"accession":"P30203","full_name":"T-cell differentiation antigen CD6","aliases":["T12","TP120"],"length_aa":668,"mass_kda":71.8,"function":"Cell adhesion molecule that mediates cell-cell contacts and regulates T-cell responses via its interaction with ALCAM/CD166 (PubMed:15048703, PubMed:15294938, PubMed:16352806, PubMed:16914752, PubMed:24584089, PubMed:24945728). Contributes to signaling cascades triggered by activation of the TCR/CD3 complex (PubMed:24584089). Functions as a costimulatory molecule; promotes T-cell activation and proliferation (PubMed:15294938, PubMed:16352806, PubMed:16914752). Contributes to the formation and maturation of the immunological synapse (PubMed:15294938, PubMed:16352806). Functions as a calcium-dependent pattern receptor that binds and aggregates both Gram-positive and Gram-negative bacteria. Binds both lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteichoic acid from Gram-positive bacteria (PubMed:17601777). LPS binding leads to the activation of signaling cascades and down-stream MAP kinases (PubMed:17601777). Mediates activation of the inflammatory response and the secretion of pro-inflammatory cytokines in response to LPS (PubMed:17601777)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P30203/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CD6","classification":"Not Classified","n_dependent_lines":20,"n_total_lines":1208,"dependency_fraction":0.016556291390728478},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CD6","total_profiled":1310},"omim":[{"mim_id":"614810","title":"MULTIPLE SCLEROSIS, SUSCEPTIBILITY TO, 5; MS5","url":"https://www.omim.org/entry/614810"},{"mim_id":"611735","title":"CUB DOMAIN-CONTAINING PROTEIN 1; CDCP1","url":"https://www.omim.org/entry/611735"},{"mim_id":"610408","title":"SOLUTE CARRIER FAMILY 15 (OLIGOPEPTIDE TRANSPORTER), MEMBER 3; SLC15A3","url":"https://www.omim.org/entry/610408"},{"mim_id":"607639","title":"SCAVENGER RECEPTOR CYSTEINE-RICH FAMILY MEMBER WITH 4 DOMAINS; SSC4D","url":"https://www.omim.org/entry/607639"},{"mim_id":"602592","title":"LYMPHOCYTE ANTIGEN CD5-LIKE; CD5L","url":"https://www.omim.org/entry/602592"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":12.6},{"tissue":"lymphoid tissue","ntpm":40.4}],"url":"https://www.proteinatlas.org/search/CD6"},"hgnc":{"alias_symbol":["Tp120"],"prev_symbol":[]},"alphafold":{"accession":"P30203","domains":[{"cath_id":"3.10.250.10","chopping":"46-101_123-156","consensus_level":"high","plddt":90.242,"start":46,"end":156},{"cath_id":"3.10.250.10","chopping":"161-260","consensus_level":"high","plddt":96.4881,"start":161,"end":260},{"cath_id":"3.10.250.10","chopping":"264-370","consensus_level":"high","plddt":94.7789,"start":264,"end":370}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P30203","model_url":"https://alphafold.ebi.ac.uk/files/AF-P30203-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P30203-F1-predicted_aligned_error_v6.png","plddt_mean":66.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD6","jax_strain_url":"https://www.jax.org/strain/search?query=CD6"},"sequence":{"accession":"P30203","fasta_url":"https://rest.uniprot.org/uniprotkb/P30203.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P30203/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P30203"}},"corpus_meta":[{"pmid":"19525953","id":"PMC_19525953","title":"Meta-analysis 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Agents.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/33287301","citation_count":26,"is_preprint":false},{"pmid":"32307907","id":"PMC_32307907","title":"Attenuation of Murine Collagen-Induced Arthritis by Targeting CD6.","date":"2020","source":"Arthritis & rheumatology (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/32307907","citation_count":26,"is_preprint":false},{"pmid":"7904289","id":"PMC_7904289","title":"Isolation and characterization of CD6- T cells from peripheral blood.","date":"1994","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/7904289","citation_count":26,"is_preprint":false},{"pmid":"32700604","id":"PMC_32700604","title":"Itolizumab, an anti-CD6 monoclonal antibody, as a potential treatment for COVID-19 complications.","date":"2020","source":"Expert opinion on biological therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32700604","citation_count":25,"is_preprint":false},{"pmid":"28289074","id":"PMC_28289074","title":"T Cell Costimulation by CD6 Is Dependent on Bivalent Binding of a GADS/SLP-76 Complex.","date":"2017","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/28289074","citation_count":25,"is_preprint":false},{"pmid":"27909060","id":"PMC_27909060","title":"CD6 Receptor Regulates Intestinal Ischemia/Reperfusion-induced Injury by Modulating Natural IgM-producing B1a Cell Self-renewal.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27909060","citation_count":25,"is_preprint":false},{"pmid":"9394826","id":"PMC_9394826","title":"Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains.","date":"1997","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/9394826","citation_count":25,"is_preprint":false},{"pmid":"10693872","id":"PMC_10693872","title":"Evidence for the expression of a second CD6 ligand by synovial fibroblasts.","date":"2000","source":"Arthritis and rheumatism","url":"https://pubmed.ncbi.nlm.nih.gov/10693872","citation_count":24,"is_preprint":false},{"pmid":"9373339","id":"PMC_9373339","title":"Production of active anti-CD6 mouse/human chimeric antibodies in the milk of transgenic mice.","date":"1995","source":"Immunotechnology : an international journal of immunological engineering","url":"https://pubmed.ncbi.nlm.nih.gov/9373339","citation_count":24,"is_preprint":false},{"pmid":"7531907","id":"PMC_7531907","title":"Analyses of monoclonal antibodies reactive with porcine CD6.","date":"1994","source":"Veterinary immunology and immunopathology","url":"https://pubmed.ncbi.nlm.nih.gov/7531907","citation_count":24,"is_preprint":false},{"pmid":"12134098","id":"PMC_12134098","title":"Risk factors for cytomegalovirus reactivation after CD6+ T-cell-depleted allogeneic bone marrow transplantation.","date":"2002","source":"Transplantation","url":"https://pubmed.ncbi.nlm.nih.gov/12134098","citation_count":23,"is_preprint":false},{"pmid":"9436462","id":"PMC_9436462","title":"CD6 dependent interactions of T cells and keratinocytes: functional evidence for a second CD6 ligand on gamma-interferon activated keratinocytes.","date":"1997","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/9436462","citation_count":22,"is_preprint":false},{"pmid":"8207228","id":"PMC_8207228","title":"Transcriptional regulation of CD6 expression on human T lymphocytes by phorbol ester.","date":"1994","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/8207228","citation_count":22,"is_preprint":false},{"pmid":"28713387","id":"PMC_28713387","title":"Human CD6 Down-Modulation following T-Cell Activation Compromises Lymphocyte Survival and Proliferative Responses.","date":"2017","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/28713387","citation_count":21,"is_preprint":false},{"pmid":"23638056","id":"PMC_23638056","title":"Fine mapping and functional analysis of the multiple sclerosis risk gene CD6.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23638056","citation_count":21,"is_preprint":false},{"pmid":"26844569","id":"PMC_26844569","title":"The Link Between CD6 and Autoimmunity: Genetic and Cellular Associations.","date":"2016","source":"Current drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/26844569","citation_count":20,"is_preprint":false},{"pmid":"7870060","id":"PMC_7870060","title":"Cloning and characterization of murine 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one","url":"https://pubmed.ncbi.nlm.nih.gov/22685579","citation_count":20,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49243,"output_tokens":9283,"usd":0.143487},"stage2":{"model":"claude-opus-4-6","input_tokens":13205,"output_tokens":3566,"usd":0.232763},"total_usd":0.37625,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"CD6 is a type I integral membrane glycoprotein with an extracellular domain containing scavenger receptor cysteine-rich (SRCR) domains, and its cytoplasmic domain contains serine residues that are substrates for phosphorylation during T cell activation.\",\n      \"method\": \"cDNA cloning, COS cell transfection, RNA blot hybridization, phosphorylation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — full-length cDNA isolation with functional expression and biochemical characterization\",\n      \"pmids\": [\"1919444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"CD6 (T12) is a 130 kDa glycoprotein on T cells that, when cross-linked with anti-CD3, delivers co-activating signals to CD4+ T cells via an IL-2-dependent pathway, and its activation is macrophage-dependent.\",\n      \"method\": \"In vitro T cell proliferation assays, IL-2R expression measurement, anti-IL-2R inhibition, anti-CD3 cross-linking\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts with defined cellular phenotype, replicated by multiple labs\",\n      \"pmids\": [\"2794503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"CD6 is a monomeric 130 kDa glycoprotein under reducing conditions with intrachain disulfide bonds, is extensively N-glycosylated, and is serine-phosphorylated in activated T cells; two anti-CD6 mAbs recognizing distinct epitopes deliver different activation signals linked to different cofactor requirements (macrophages vs. PMA).\",\n      \"method\": \"Surface radiolabeling, immunoprecipitation, Western blot, biosynthetic labeling with tunicamycin, phosphorylation assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple biochemical methods characterizing structure and post-translational modifications\",\n      \"pmids\": [\"2481822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"CD6 surface expression is regulated by phosphorylation: PKC activation rapidly converts an unphosphorylated 105 kDa form to a phosphorylated 130 kDa form without requiring new protein synthesis; this interconversion is reversible and alkaline phosphatase treatment converts the 130 kDa form back to 105 kDa.\",\n      \"method\": \"Surface 125I labeling, immunoprecipitation, 32P labeling, alkaline phosphatase treatment, PKC activator stimulation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical demonstration of reversible phosphorylation-dependent isoform conversion\",\n      \"pmids\": [\"2384666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"CD6 biosynthesis proceeds through an 88 kDa nascent polypeptide, an immature N-glycosylated 110 kDa intermediate, and a mature 130 kDa surface form bearing sulfated O-linked oligosaccharides; CD6 is phosphorylated in resting cells and can be hyperphosphorylated by PKC activators.\",\n      \"method\": \"Pulse-chase biosynthetic labeling, tunicamycin treatment, 32P labeling, immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — detailed biosynthetic characterization with multiple orthogonal biochemical methods\",\n      \"pmids\": [\"2016320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"CD6 cytoplasmic tyrosine residues become phosphorylated upon TCR/CD3 stimulation; co-crosslinking with CD4 produces the highest level of CD6 tyrosine phosphorylation, while co-crosslinking with CD2 also augments it, but crosslinking CD2, CD4, or CD28 alone does not phosphorylate CD6.\",\n      \"method\": \"Anti-phosphotyrosine immunoprecipitation, co-crosslinking of CD3 with CD2, CD4, or CD28 on primary T cells\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical demonstration of TCR-dependent tyrosine phosphorylation with defined cofactor requirements\",\n      \"pmids\": [\"7678115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"CD6 has a large cytoplasmic domain of 244 amino acids (previously underestimated) containing two proline-rich SH3-binding motifs, serine-threonine-rich repeats, PKC phosphorylation sites, and casein kinase-2 sites; alternatively spliced cytoplasmic isoforms exist in human peripheral blood lymphocytes.\",\n      \"method\": \"RT-PCR, cDNA cloning and sequencing, COS cell transfection, immunoprecipitation, SDS-PAGE\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — sequence-based domain identification validated by functional expression\",\n      \"pmids\": [\"7589069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"ALCAM (CD166) is a CD6 ligand: COS cells expressing CD6 adhere to thymic epithelial cells, this adhesion is blocked by anti-CD6 mAb, and an ALCAM-Rg fusion protein binds specifically to CD6 transfectants; ALCAM-CD6 interaction mediates thymocyte–thymic epithelial cell adhesion.\",\n      \"method\": \"COS cell transfection, cell adhesion assays, antibody blocking, immunoglobulin fusion protein binding, cDNA cloning\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted receptor-ligand interaction with reciprocal binding and functional adhesion assay\",\n      \"pmids\": [\"7760007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The membrane-proximal SRCR domain (domain 3) of CD6 contains the ALCAM binding site; mAbs binding to this domain preferentially block CD6-ALCAM binding.\",\n      \"method\": \"Domain-specific CD6-Rg fusion proteins, cell adhesion assays, antibody blocking experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic domain-deletion fusion protein analysis with functional adhesion readout\",\n      \"pmids\": [\"7543097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The amino-terminal Ig-like domain of ALCAM specifically binds the membrane-proximal (third) SRCR domain of CD6 in a 1:1 stoichiometry, representing the first characterized Ig-domain/SRCR-domain protein interaction.\",\n      \"method\": \"Truncated Ig fusion proteins, receptor-ligand binding assays, thrombin cleavage to produce monomeric domains, stoichiometry determination\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted interaction with defined stoichiometry using truncated recombinant proteins\",\n      \"pmids\": [\"8663238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Three residues in the membrane-proximal SRCR domain (domain 3) of CD6, located in a region of low sequence conservation, when mutated abolish ALCAM binding without disrupting overall CD6 conformation (as shown by intact mAb binding); these are the first residues critical for SRCR domain-ligand interaction.\",\n      \"method\": \"Site-directed mutagenesis, binding assays with anti-CD6 mAbs, ALCAM binding assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with orthogonal structural controls identifies specific binding residues\",\n      \"pmids\": [\"9054570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Residues critical for CD6 binding in ALCAM cluster on the predicted A'GFCC'C\\\" face of ALCAM's N-terminal Ig domain, identified by targeted mutagenesis; this site is conserved across species.\",\n      \"method\": \"Targeted mutagenesis of ALCAM, CD6-ALCAM binding assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis-based mapping of binding site with structural modeling\",\n      \"pmids\": [\"8823162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD6 interacts with CD166 (ALCAM) with a KD of 0.4–1.0 μM and fast off-rate; soluble monomeric CD6 or CD166 at concentrations blocking this interaction inhibit antigen-specific human T cell responses, demonstrating that extracellular CD6-CD166 engagement is required for optimal immune response.\",\n      \"method\": \"Surface plasmon resonance (SPR) kinetics, soluble protein inhibition of antigen-specific T cell proliferation assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative binding kinetics plus functional inhibition with defined concentrations\",\n      \"pmids\": [\"15048703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CD6 gene is located on chromosome 11q13 near CD5 and CD20; it is encoded by at least 13 exons with each SRCR domain encoded by a separate exon; at least five cytoplasmic domain isoforms exist from alternative splicing of cytoplasmic exons.\",\n      \"method\": \"Genomic cloning, FISH, YAC library screening, RT-PCR, mRNA analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — genomic characterization with direct structural evidence for splice isoforms\",\n      \"pmids\": [\"9013954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CD6 cytoplasmic tyrosine residues Y629 and Y662 (the two C-terminal tyrosines) are critical for TCR-induced tyrosine phosphorylation; isoform CD6e lacking proline-rich motifs is not phosphorylated; Ca2+ mobilization by CD6/TCR co-ligation requires a membrane-proximal cytoplasmic region N-terminal to amino acid 555, distinct from the tyrosine phosphorylation region.\",\n      \"method\": \"Chimeric receptor expression, TCR cross-linking, anti-phosphotyrosine immunoprecipitation, Ca2+ flux measurement, site-directed mutagenesis\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis of specific residues with functional phosphorylation and calcium flux readouts\",\n      \"pmids\": [\"9394826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD6 physically associates with the TCR/CD3 complex (shown by co-immunoprecipitation, co-capping, and FRET); CD6 and ALCAM colocalize with TCR/CD3 at the central SMAC of the immunological synapse; soluble CD6 reduces T cell-APC conjugate formation and inhibits CD3-mediated T cell proliferation in a dose-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, co-capping, FRET, confocal microscopy of Ag-specific conjugates, proliferation inhibition assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods demonstrating physical association and immunological synapse localization with functional consequence\",\n      \"pmids\": [\"15294938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CD5 and CD6 physically associate at the lymphocyte cell membrane, co-immunoprecipitate from Brij 96 detergent lysates independently of other surface receptors, and co-accumulate at the immunological synapse; this association is independent of the CD5 cytoplasmic region.\",\n      \"method\": \"Co-immunoprecipitation, FRET, co-capping, co-modulation experiments, imaging of T cell-APC conjugates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus FRET and co-capping demonstrating physical in vivo association\",\n      \"pmids\": [\"12473675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD6 (rat OX52) co-precipitates with CD5, and the CD5 fraction associated with CD6 is highly phosphorylated compared to CD5 associated with TCR or CD2; CD6 uniquely associates with Lck, Fyn, ZAP-70, and the Tec-family kinase Itk (not found in CD2/CD5/TCR complexes); Lck+Itk effectively phosphorylate CD5 cytoplasmic peptides.\",\n      \"method\": \"Immunoprecipitation, in vitro kinase assays with synthetic peptides, RT-PCR cloning of rat CD6\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP with kinase assays identifying unique Itk association and CD5 phosphorylation role\",\n      \"pmids\": [\"12525577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CD6 and ALCAM are actively recruited to the DC-T cell contact zone; ALCAM-blocking antibodies interfere with DC-T cell conjugate formation; CD6-ALCAM interactions are required during the proliferative phase of T cell response; CD6 cross-linking with CD3 enhances proliferation to levels similar to CD28 co-stimulation.\",\n      \"method\": \"Fluorescence microscopy of DC-T cell contacts, antibody blocking of conjugate formation, T cell proliferation assays with blocking antibodies and recombinant ALCAM-Fc, CD6/CD3 co-crosslinking\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays demonstrating adhesion and costimulatory roles with defined mechanistic interventions\",\n      \"pmids\": [\"16352806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The cytoplasmic tail of CD6 interacts with syntenin-1, a PDZ domain-containing scaffolding protein, through CD6's C-terminal sequence (-YDDISAA) and both PDZ domains of syntenin-1; syntenin-1 accumulates at CD6 caps and at the immunological synapse.\",\n      \"method\": \"Yeast two-hybrid screen, mutational analysis, pull-down assays, co-immunoprecipitation in mammalian cells, confocal microscopy\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid discovery confirmed by pull-down, co-IP, and mutagenesis in mammalian cells\",\n      \"pmids\": [\"16034076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD6 ligation recruits the adaptor SLP-76 through phosphorylation-dependent binding to tyrosine 662 (Y662) in the CD6 cytoplasmic domain (KD = 0.5 μM at 37°C); this interaction mediates CD6 costimulation; Y662F mutation abolishes both SLP-76 binding and costimulation; CD6 and SLP-76 co-precipitate from normal human T cells.\",\n      \"method\": \"Phosphopeptide binding assay (SPR/equilibrium), co-precipitation from primary T cells, CD6 cytoplasmic mutant analysis in T cell hybridoma model\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct binding kinetics of phosphopeptide to SLP-76 plus mutagenesis and co-IP in primary cells\",\n      \"pmids\": [\"16914752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD6 ligation (by mAbs or ALCAM-Fc) induces time- and dose-dependent activation of ERK1/2, p38, and JNK MAPK cascades in T cells; this requires the C-terminal cytoplasmic region of CD6 and Src tyrosine kinases; CD6 ligation activates AP-1 and c-Fos SRE transcription; synergistic effects occur with TCR/CD3 co-ligation.\",\n      \"method\": \"Western blot for phospho-MAPK, reporter gene assays (SRE-luciferase, AP-1-luciferase), Src kinase inhibitors, mAb and ALCAM-Fc stimulation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple MAPK pathways confirmed with pharmacological inhibitors and reporter assays\",\n      \"pmids\": [\"16818773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A CD6 isoform lacking SRCR domain 3 (CD6Δd3), generated by exon 5 skipping, cannot localize to the immunological synapse (unlike full-length CD6), demonstrating that SRCR domain 3 (the ALCAM-binding domain) is required for CD6 targeting to the T cell-APC interface; CD6Δd3 is upregulated upon T cell activation.\",\n      \"method\": \"cDNA cloning, flow cytometry, confocal imaging of immunological synapse, RT-PCR at single-cell level, Western blot\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging of IS localization with domain deletion mutant and multiple expression analyses\",\n      \"pmids\": [\"17371992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CD6 binds lipoteichoic acid (Gram-positive bacteria) and LPS (Gram-negative bacteria) through its ectodomain; the KD for LPS-CD6 interaction is ~2.69×10⁻⁸ M; membrane CD6 also retains LPS-binding ability and activates the MAPK signaling cascade upon LPS binding; recombinant soluble CD6 protects mice from lethal LPS challenge and reduces TNF-α, IL-6, and IL-1β.\",\n      \"method\": \"Binding assays with recombinant soluble CD6, bacterial aggregation, SPR/affinity measurement, MAPK activation assays, in vivo LPS challenge mouse model, serum cytokine measurement\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding with quantified affinity plus in vivo functional protection, multiple methods\",\n      \"pmids\": [\"17601777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CD6 significantly attenuates early and late TCR signaling: expression of CD6 (but not a cytoplasmic domain deletion mutant) reduces calcium mobilization and IL-2 release after superantigen or anti-CD3 stimulation; morpholino-mediated knockdown of CD6 enhances calcium signals; blocking CD6-CD166 interaction with anti-CD166 increases T cell proliferation, but anti-CD6 antibodies inhibit it.\",\n      \"method\": \"Single-cell calcium imaging, IL-2 ELISA, Jurkat overexpression of CD6 vs. cytoplasmic deletion mutant, morpholino knockdown in primary human T cells, antibody blocking proliferation assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple complementary approaches (overexpression, KD, deletion mutant) with early and late signaling readouts\",\n      \"pmids\": [\"21956609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Quantitative mass spectrometry of primary mouse T cells revealed that CD6 recruits SLP-76 and Vav1 independently of the LAT adaptor, constituting a LAT-independent TCR signaling hub; 112 high-confidence time-resolved protein interactions around Zap70, Lat, and SLP-76 were mapped.\",\n      \"method\": \"Knock-in affinity tag mass spectrometry (quantitative proteomics) in primary CD4+ T cells, time-resolved interaction mapping\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — knock-in quantitative MS with time resolution in primary cells, identifies LAT-independent CD6-SLP-76-Vav1 pathway\",\n      \"pmids\": [\"24584089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"X-ray crystal structures of CD6 (three SRCR domains) and CD166 (two N-terminal domains) reveal a nonlinear organization of consecutive SRCR domains; a disease-associated SNP in CD6 introduces a glycosylation site that sterically hinders the CD6/CD166 interaction; native MS shows competition between heterophilic CD6-CD166 and homophilic CD166-CD166 interactions.\",\n      \"method\": \"X-ray crystallography, native mass spectrometry, glycosylation site mutagenesis, binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus native MS and mutagenesis providing mechanistic insight into SNP effect\",\n      \"pmids\": [\"26146185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ALCAM recruitment to adhesion sites and its membrane tether-anchoring propensity depend on actin cytoskeletal interactions; linking ALCAM to the actin cortex strengthens CD6-mediated cell adhesion and stiffens the cortex, forming a mechanical link between CD6 (on T cells) and the actin cortex (via ALCAM on DCs) at the immunological synapse.\",\n      \"method\": \"Single-cell force spectroscopy, TIRF microscopy, ALCAM cytoplasmic tail mutation constructs, actin cortex stiffness measurements\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted adhesion mechanics with cytoplasmic domain mutants and biophysical measurements\",\n      \"pmids\": [\"24496453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"T cell activation regulates CD6 alternative splicing (exon 5 skipping to produce CD6Δd3) through increased RNA Pol II occupancy and chromatin acetylation; the splicing factor SRSF1 binds CD6 intron 4 to promote exon 5 inclusion, and upon activation SRSF1 is downregulated and its recruitment to CD6 transcript is impaired by increased chromatin acetylation.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for RNA Pol II and acetylation, RNA immunoprecipitation (RIP) for SRSF1, histone deacetylase inhibitor experiments, RT-PCR\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — ChIP and RIP with mechanistic intervention (HDAC inhibitor) identifying SRSF1 as splicing regulator\",\n      \"pmids\": [\"24890719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD6 cytoplasmic Y629 residue recruits the SH2 adaptor GADS; bivalent recruitment of a GADS/SLP-76 complex (at Y629 and Y662 respectively) is required for CD6 costimulation; both Y629F and Y662F mutations abolish costimulation in Jurkat and primary T cells.\",\n      \"method\": \"Biochemical pulldown, mutational analysis of Y629F and Y662F in Jurkat and primary T cells, T cell activation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with functional costimulation readout identifying bivalent GADS/SLP-76 recruitment mechanism\",\n      \"pmids\": [\"28289074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD318 is a CD6 ligand distinct from CD166: identified using mAb 3A11 immunoprecipitation and proteomics; CD318 KO mice are protected from experimental autoimmune encephalomyelitis (like CD6 KO mice); CD318 mediates CD6-dependent adhesion of T cells to synovial fibroblasts.\",\n      \"method\": \"Mass spectrometry protein identification, CD318 KO mouse EAE model, T cell adhesion assays to synovial fibroblasts, soluble CD318 chemotaxis assay\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS identification of ligand confirmed by KO phenotype and functional adhesion assays\",\n      \"pmids\": [\"28760953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRISPR/Cas9-based quantitative MS of primary mouse T cells defines the CD6 signalosome as comprising both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation; CD6 also associates constitutively (independently of TCR engagement) with proteins supporting T cell transendothelial migration.\",\n      \"method\": \"CRISPR/Cas9 knock-in in primary mouse T cells, quantitative mass spectrometry, TCR stimulation time-course\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — primary cell CRISPR knock-in with quantitative MS identifying dual positive/negative signalosome composition\",\n      \"pmids\": [\"33125054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CD6 deficiency in mice reduces CD4+ and CD8+ single-positive thymocyte numbers; CD6−/− double-positive thymocytes show increased Ca2+ mobilization to TCR cross-linking; bone marrow chimera experiments reveal a T cell-autonomous selective disadvantage of CD6−/− cells during development; CD6−/− mice show increased effector/memory and regulatory T cells but diminished Treg suppressive activity.\",\n      \"method\": \"CD6 KO mouse analysis, bone marrow chimeras, TCR-transgenic crosses, Ca2+ flux assays, Treg suppression assays, collagen-induced arthritis model\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — comprehensive in vivo KO analysis with bone marrow chimeras and multiple T cell subset phenotyping\",\n      \"pmids\": [\"27377588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD6 KO mice show decreased pathogenic T cell responses, reduced spinal cord T cell infiltration, and attenuated EAE; CD6-deficient T cells show augmented initial activation but reduced survival and proliferation, leading to decreased Th1 and Th17 polarization; activated CD6-deficient T cells show impaired infiltration through brain microvascular endothelial cell monolayers.\",\n      \"method\": \"CD6 KO mouse EAE model, T cell recall assays, Th1/Th17 polarization assays, transendothelial migration assay, CD6-humanized mouse treatment with anti-human CD6 mAb\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple defined functional phenotypes plus humanized mouse treatment validation\",\n      \"pmids\": [\"28209777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CD6 ligation protects chronic lymphocytic leukemia B cells from anti-IgM-induced apoptosis by downregulating bax-α mRNA levels and preventing bcl-2 downregulation, resulting in an increased Bcl-2/Bax ratio.\",\n      \"method\": \"Anti-CD6 crosslinking on B-CLL cells, Northern blot for bax-α and bcl-2 mRNA, apoptosis assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct mechanistic link from CD6 ligation to apoptosis resistance via Bcl-2/Bax ratio, single lab\",\n      \"pmids\": [\"9108402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD6 and CD166/ALCAM interact with Galectin-1 and Galectin-3 in a carbohydrate-dependent manner; galectins interfere with superantigen-induced T cell proliferation and CD6-CD166-mediated cell adhesion; CD6 expression protects cells from galectin-induced apoptosis.\",\n      \"method\": \"Galectin binding assays, T cell proliferation assays with galectin addition, cell adhesion assays, apoptosis measurement\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — binding and functional assays in single study without detailed mechanistic dissection\",\n      \"pmids\": [\"24945728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CD6 is selectively expressed on B1 cells outside the bone marrow and peritoneal cavity; CD6−/− mice are protected from intestinal ischemia/reperfusion injury, with reduced natural IgM titers and B1a cell populations, demonstrating a role for CD6 in B1a cell self-renewal.\",\n      \"method\": \"CD6 KO mouse intestinal I/R model, flow cytometry for B1a cells, serum natural IgM ELISA\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with mechanistic link to B1a self-renewal and IgM production, single study\",\n      \"pmids\": [\"27909060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"PKC activation by phorbol ester (PMA) increases CD6 surface expression by increasing CD6 mRNA transcription (nuclear run-on); this requires new protein synthesis; CD2 ligation (but not ligation of other surface molecules tested) upregulates CD6 expression on thymocytes but not mature T cells.\",\n      \"method\": \"Northern blot, nuclear run-on transcription assays, Western blot, flow cytometry, PKC inhibitor studies, actinomycin D and cycloheximide experiments\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — nuclear run-on directly measures transcription rate; multiple controls including translation inhibitors\",\n      \"pmids\": [\"8207228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"A second CD6 ligand (3A11 antigen, later identified as CD318) distinct from CD166 is expressed on cells from thymus, skin, synovium, and cartilage; its expression is enhanced by IFN-γ; it has a molecular mass of 130 kDa and is immunoprecipitated by soluble CD6-Ig fusion protein; anti-CD166 siRNA knockdown does not alter 3A11 expression.\",\n      \"method\": \"Immunoprecipitation with CD6-Ig fusion protein, confocal microscopy, siRNA knockdown of CD166, flow cytometry, T cell adhesion assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — distinct ligand confirmed by reciprocal immunoprecipitation and siRNA exclusion of CD166, single lab\",\n      \"pmids\": [\"15528349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Itolizumab (anti-CD6 D1 mAb) directly inhibits CD6 receptor hyperphosphorylation and decreases associated ZAP70 kinase and SLP76 docking protein levels; F(ab')2 fragment of Itolizumab does not produce this inhibition, indicating the intact antibody format is required for the mechanism.\",\n      \"method\": \"CD6 phosphorylation Western blot, ZAP70 and SLP76 co-immunoprecipitation, Itolizumab vs. F(ab')2 comparison\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical measurement of phosphorylation and signaling complex, single lab\",\n      \"pmids\": [\"28672038\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD6 is a type I transmembrane SRCR superfamily glycoprotein expressed primarily on T cells that functions as a multitask signaling hub at the immunological synapse: its membrane-proximal SRCR domain 3 binds ligands CD166/ALCAM and CD318 (and pathogen-associated patterns including LPS), recruiting CD6 to the TCR/CD3 complex at the central SMAC; TCR activation induces tyrosine phosphorylation of cytoplasmic residues Y629 and Y662, enabling bivalent recruitment of a GADS/SLP-76 complex (costimulatory) and independently of LAT recruits Vav1, while also associating with the negative regulator UBASH3A/STS-2, making CD6 both a positive and negative rheostat of T cell activation; alternative splicing of exon 5 (regulated by SRSF1 and chromatin acetylation upon activation) produces a CD6Δd3 isoform that lacks the ALCAM-binding domain and cannot localize to the immunological synapse, providing a mechanism to tune CD6-mediated signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CD6 is a type I transmembrane glycoprotein of the scavenger receptor cysteine-rich (SRCR) superfamily that functions as a dual costimulatory and inhibitory signaling hub at the T cell immunological synapse, integrating adhesion, pathogen sensing, and TCR signal modulation. Its membrane-proximal SRCR domain 3 binds the ligands CD166/ALCAM (KD ~0.4–1.0 µM) and CD318, mediating T cell–APC adhesion and targeting CD6 to the central SMAC where it physically associates with the TCR/CD3 complex and CD5 [PMID:7760007, PMID:15294938, PMID:28760953, PMID:12473675]. TCR engagement triggers tyrosine phosphorylation of cytoplasmic residues Y629 and Y662, which bivalently recruit a GADS/SLP-76 signalosome independently of LAT, while also engaging the negative regulator UBASH3A/STS-2, thereby enabling CD6 to both promote and attenuate T cell activation depending on context; CD6 deficiency in mice causes augmented initial TCR signaling but impaired T cell survival, reduced Th1/Th17 responses, and protection from EAE [PMID:28289074, PMID:24584089, PMID:33125054, PMID:27377588, PMID:28209777]. T cell activation induces alternative splicing of exon 5 (regulated by SRSF1 and chromatin acetylation) to produce a CD6Δd3 isoform lacking the ALCAM-binding domain that cannot localize to the immunological synapse, providing a feedback mechanism to tune CD6-mediated costimulation [PMID:17371992, PMID:24890719].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Establishing CD6 as a T cell costimulatory molecule: prior to this, CD6 (T12) was an orphan 130 kDa T cell surface glycoprotein; cross-linking with anti-CD3 demonstrated that it delivers IL-2-dependent co-activating signals to CD4+ T cells, requiring macrophage cofactors.\",\n      \"evidence\": \"In vitro T cell proliferation and IL-2R expression assays with anti-CD6/anti-CD3 co-crosslinking\",\n      \"pmids\": [\"2794503\", \"2481822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Costimulatory ligand unknown\", \"Signaling pathway downstream of CD6 uncharacterized\", \"Contribution of macrophages mechanistically undefined\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Molecular cloning and biochemical characterization revealed that CD6 is an SRCR superfamily member with extracellular SRCR domains and a cytoplasmic domain subject to serine phosphorylation and PKC-regulated electrophoretic mobility shifts, establishing its signaling potential.\",\n      \"evidence\": \"cDNA cloning, COS cell transfection, pulse-chase biosynthetic labeling, reversible phosphatase treatment\",\n      \"pmids\": [\"1919444\", \"2384666\", \"2016320\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tyrosine phosphorylation not yet examined\", \"Ligand not identified\", \"Relationship to TCR signaling unknown\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of ALCAM/CD166 as the CD6 ligand and mapping the interaction to SRCR domain 3 resolved the long-standing question of what CD6 binds, establishing this as the first characterized SRCR–Ig-domain receptor–ligand pair.\",\n      \"evidence\": \"COS cell adhesion assays, Ig fusion protein binding, domain-deletion constructs, mAb blocking\",\n      \"pmids\": [\"7760007\", \"7543097\", \"8663238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding affinity not yet quantified\", \"In vivo relevance of CD6-ALCAM interaction unproven\", \"Additional ligands suspected but not identified\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Identification of Y629 and Y662 as the TCR-induced phosphorylation sites in the CD6 cytoplasmic domain, and demonstration that calcium mobilization maps to a distinct membrane-proximal region, established that CD6 signaling involves separable functional modules.\",\n      \"evidence\": \"Site-directed mutagenesis of chimeric receptors, anti-phosphotyrosine IP, calcium flux measurement in T cell lines\",\n      \"pmids\": [\"9394826\", \"7678115\", \"9013954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream adaptor proteins binding phosphotyrosines unknown\", \"In vivo significance of individual tyrosines untested\", \"Contribution of alternatively spliced cytoplasmic isoforms unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Quantitative kinetic measurement of CD6–ALCAM interaction (KD 0.4–1.0 µM, fast off-rate) and demonstration that CD6 physically associates with TCR/CD3 at the central SMAC placed CD6 as an integral component of the immunological synapse required for optimal T cell responses.\",\n      \"evidence\": \"Surface plasmon resonance, co-immunoprecipitation, FRET, confocal microscopy of antigen-specific conjugates, soluble protein inhibition of proliferation\",\n      \"pmids\": [\"15048703\", \"15294938\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of CD6 recruitment to TCR complex unknown\", \"Role of second ligand (3A11/CD318) at IS undefined\", \"Structural basis of CD6-TCR association unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that phospho-Y662 recruits SLP-76 directly and that CD6 ligation activates ERK1/2, p38, JNK, and AP-1 pathways identified the core costimulatory signaling cascade downstream of CD6.\",\n      \"evidence\": \"Phosphopeptide–SLP-76 SPR binding, Y662F mutagenesis abolishing costimulation, phospho-MAPK Western blots, reporter gene assays with Src kinase inhibitors\",\n      \"pmids\": [\"16914752\", \"16818773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SLP-76 recruitment to CD6 is LAT-dependent or independent not resolved\", \"Identity of adaptor binding Y629 unknown\", \"MAPK activation mechanism via CD6 vs. TCR not dissected\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovery of the CD6Δd3 splice isoform (lacking exon 5/SRCR domain 3) that cannot localize to the immunological synapse and is upregulated upon T cell activation established a built-in feedback mechanism to modulate CD6-mediated costimulation.\",\n      \"evidence\": \"RT-PCR, confocal imaging of IS localization with domain-deleted construct, single-cell splice variant analysis\",\n      \"pmids\": [\"17371992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Splicing regulation mechanism unknown\", \"Functional consequence of CD6Δd3 upregulation on T cell effector function untested\", \"Whether CD6Δd3 acts as dominant negative unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstration that CD6 ectodomain binds bacterial LPS and lipoteichoic acid with nanomolar affinity and that soluble CD6 protects mice from lethal endotoxemia revealed an innate immune pattern-recognition function for CD6 beyond adaptive T cell costimulation.\",\n      \"evidence\": \"Recombinant soluble CD6 binding assays, SPR affinity measurement (KD ~27 nM for LPS), in vivo LPS challenge with survival and cytokine readouts\",\n      \"pmids\": [\"17601777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which SRCR domain binds LPS not mapped\", \"Signaling pathway triggered by LPS–CD6 on T cells not dissected separately from TCR signals\", \"Physiological relevance of CD6 pathogen sensing in infection models untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Quantitative proteomics in primary T cells revealed that CD6 recruits SLP-76 and Vav1 independently of the canonical adaptor LAT, establishing CD6 as a LAT-independent TCR signaling node, while concurrent work identified SRSF1 and chromatin acetylation as regulators of CD6 exon 5 alternative splicing.\",\n      \"evidence\": \"Knock-in affinity-tag MS in primary mouse CD4+ T cells; ChIP for Pol II/acetylation and RIP for SRSF1 with HDAC inhibitor perturbation\",\n      \"pmids\": [\"24584089\", \"24890719\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How GADS fits into the LAT-independent pathway not yet shown\", \"Whether Vav1 recruitment is direct or SLP-76-mediated unclear\", \"Epigenetic regulation of CD6 splicing in disease states untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"CD6 knockout mouse analysis revealed that CD6 deficiency causes augmented initial TCR calcium signaling but reduced thymocyte numbers and impaired Treg function, establishing CD6 as a dual positive/negative modulator of T cell development and activation in vivo.\",\n      \"evidence\": \"CD6 KO mice with bone marrow chimeras, TCR-transgenic crosses, calcium flux, Treg suppression assays, collagen-induced arthritis model\",\n      \"pmids\": [\"27377588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of enhanced calcium signaling in CD6-deficient cells undefined\", \"Mechanism of impaired Treg function unclear\", \"Whether phenotype is ALCAM-dependent or CD318-dependent not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of CD318 as a second CD6 ligand, demonstration that bivalent GADS(Y629)/SLP-76(Y662) recruitment is required for costimulation, and CD6 KO protection from EAE with impaired T cell transendothelial migration collectively established CD6 as a multi-ligand receptor integrating costimulation with tissue infiltration.\",\n      \"evidence\": \"MS-based CD318 identification, CD318 KO EAE model, Y629F/Y662F mutagenesis in Jurkat and primary T cells, brain endothelial transmigration assays\",\n      \"pmids\": [\"28760953\", \"28289074\", \"28209777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which CD6 domain binds CD318 not mapped\", \"Relative contributions of ALCAM vs. CD318 to IS formation undefined\", \"Transendothelial migration mechanism downstream of CD6 not molecularly dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"CRISPR knock-in quantitative MS defined the full CD6 signalosome as containing both positive regulators (SLP-76, ZAP70, VAV1) and the negative regulator UBASH3A/STS-2, plus constitutive associations supporting transendothelial migration, resolving the paradox of CD6 serving as both costimulatory and inhibitory.\",\n      \"evidence\": \"CRISPR/Cas9 knock-in in primary mouse T cells with time-resolved quantitative MS after TCR stimulation\",\n      \"pmids\": [\"33125054\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which UBASH3A counteracts CD6 costimulation unknown\", \"Stoichiometry of positive vs. negative regulator recruitment not determined\", \"Whether constitutive CD6 associations change during inflammation not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how UBASH3A-mediated negative regulation mechanistically opposes GADS/SLP-76 costimulation; which SRCR domain(s) bind CD318; how CD6 dual signaling is partitioned during thymic selection versus peripheral activation; and whether CD6Δd3 actively suppresses signaling or merely fails to engage the synapse.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"UBASH3A inhibitory mechanism via CD6 uncharacterized\", \"CD318 binding domain on CD6 unmapped\", \"No structural model of full-length CD6 cytoplasmic domain with adaptors\", \"Functional role of CD6Δd3 beyond failure to localize is untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [20, 29, 31]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [24, 32, 33]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [7, 12, 18, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 7, 15, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [1, 21, 25, 29]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 18, 24, 31, 32, 33]}\n    ],\n    \"complexes\": [\n      \"TCR/CD3 complex (recruited upon activation)\",\n      \"CD5-CD6 complex\"\n    ],\n    \"partners\": [\n      \"ALCAM\",\n      \"CDCP1\",\n      \"SLP76\",\n      \"GADS\",\n      \"VAV1\",\n      \"UBASH3A\",\n      \"CD5\",\n      \"SDCBP\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}