RESUMO
Inflammasomes are important sentinels of innate immune defence, sensing pathogens and inducing cell death in infected cells1. There are several inflammasome sensors that each detect and respond to a specific pathogen- or damage-associated molecular pattern (PAMP or DAMP, respectively)1. During infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which can simultaneously engage multiple inflammasome sensors2-5. Here we found that AIM2 regulates the innate immune sensors pyrin and ZBP1 to drive inflammatory signalling and a form of inflammatory cell death known as PANoptosis, and provide host protection during infections with herpes simplex virus 1 and Francisella novicida. We also observed that AIM2, pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD, that drove inflammatory cell death (PANoptosis). Collectively, our findings define a previously unknown regulatory and molecular interaction between AIM2, pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that we term the AIM2 PANoptosome and comprising multiple inflammasome sensors and cell death regulators. These results advance the understanding of the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and pyrin-mediated diseases.
Assuntos
Apoptose/imunologia , Proteínas de Ligação a DNA/metabolismo , Necroptose/imunologia , Pirina/metabolismo , Piroptose/imunologia , Proteínas de Ligação a RNA/metabolismo , Animais , Caspase 1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Feminino , Francisella , Herpesvirus Humano 1 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células THP-1RESUMO
Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.
Assuntos
Aspergilose/prevenção & controle , Aspergillus fumigatus/metabolismo , Inflamassomos/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Polissacarídeos/metabolismo , Animais , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Biofilmes , Colite/induzido quimicamente , Colite/prevenção & controle , Sulfato de Dextrana , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Imunidade Inata , Inflamassomos/imunologia , Masculino , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polissacarídeos/imunologia , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismoRESUMO
The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.
Assuntos
Ácido Oleico , Peptídeos , Staphylococcus aureus , Microscopia Crioeletrônica , Ácidos Graxos Insaturados , Bicamadas Lipídicas/metabolismo , Fosfatos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyAâ¢membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.
RESUMO
The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.
Assuntos
Receptores de Citocinas/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Domínios RING Finger , Receptores do Fator Autócrino de Motilidade , Receptores de Citocinas/metabolismo , Receptores de Citocinas/fisiologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , UbiquitinaçãoRESUMO
Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 µM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.
Assuntos
Antígenos CD1/metabolismo , Lipopeptídeos/metabolismo , Oxazóis/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linhagem Celular , Células HEK293 , Humanos , Lipopeptídeos/imunologia , Ativação Linfocitária/imunologia , Oxazóis/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Tuberculose/imunologiaRESUMO
Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana , Imagem Individual de Molécula , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Humanos , AnimaisRESUMO
Type I interferons (IFNs) are essential innate immune proteins that maintain tissue homeostasis through tonic expression and can be upregulated to drive antiviral resistance and inflammation upon stimulation. However, the mechanisms that inhibit aberrant IFN upregulation in homeostasis and the impacts of tonic IFN production on health and disease remain enigmatic. Here, we report that caspase-8 negatively regulates type I IFN production by inhibiting the RIPK1-TBK1 axis during homeostasis across multiple cell types and tissues. When caspase-8 is deleted or inhibited, RIPK1 interacts with TBK1 to drive elevated IFN production, leading to heightened resistance to norovirus infection in macrophages but also early onset lymphadenopathy in mice. Combined deletion of caspase-8 and RIPK1 reduces the type I IFN signaling and lymphadenopathy, highlighting the critical role of RIPK1 in this process. Overall, our study identifies a mechanism to constrain tonic type I IFN during homeostasis which could be targeted for infectious and inflammatory diseases.
Assuntos
Interferon Tipo I , Linfadenopatia , Animais , Antivirais , Caspase 8 , Homeostase , Camundongos , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.
Assuntos
Quimiocina CCL22 , Células Matadoras Naturais , Transtornos Linfoproliferativos , Animais , Quimiocina CCL22/genética , Células Matadoras Naturais/patologia , Ativação Linfocitária , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Transtornos Linfoproliferativos/patologia , Camundongos , MutaçãoRESUMO
Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.
Assuntos
Microscopia Crioeletrônica , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/ultraestrutura , Sítios de Ligação , Biocatálise , Domínio Catalítico , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Mutação , Reprodutibilidade dos Testes , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/genética , Especificidade por SubstratoRESUMO
Heparanase, an endo-ß-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains and plays important roles in cellular growth and metastasis. Heparanase assays reported to-date are labor intensive, complex and/or expensive. A simpler assay is critically needed to understand the myriad roles of heparanase. We reasoned that fluorescent heparin could serve as an effective probe of heparanase levels. Following synthesis and screening, a heparin preparation labeled with DABCYL and EDANS was identified, which exhibited a characteristic increase in signal following cleavage by human heparanase. This work describes the synthesis of this heparin substrate, its kinetic and spectrofluorometric properties, optimization of the heparanase assay, use of the assay in inhibitor screening, and elucidation of the state of heparanase in different cell lines. Our FRET-based assay is much simpler and more robust than all assays reported in the literature as well as a commercially available kit.
Assuntos
Corantes Fluorescentes/química , Glucuronidase/química , Heparina/análogos & derivados , Heparina/química , Naftalenossulfonatos/química , p-Dimetilaminoazobenzeno/análogos & derivados , Animais , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Heparina/síntese química , Humanos , Células MCF-7 , Naftalenossulfonatos/síntese química , Células Sf9 , Spodoptera , p-Dimetilaminoazobenzeno/síntese química , p-Dimetilaminoazobenzeno/químicaRESUMO
Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P4, I(1,3,4,5,6)P5, and IP6 promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP6 and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP6 production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100-600 µM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL's activating switch in NED triggered by a select repertoire of IP metabolites.
Assuntos
Fosfatos de Inositol/metabolismo , Proteínas Quinases/metabolismo , Animais , Sobrevivência Celular , Células HT29 , Humanos , Proteínas Quinases/isolamento & purificação , Células Sf9 , SpodopteraRESUMO
Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Compostos Azo/química , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Óxido Nítrico/metabolismo , Piperazinas/química , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/química , Compostos Azo/síntese química , Compostos Azo/farmacologia , Linhagem Celular Tumoral , Células HL-60 , Humanos , Camundongos , Doadores de Óxido Nítrico , Piperazinas/síntese química , Piperazinas/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Células U937RESUMO
Mutations in the gene BEST1 are causally associated with as many as five clinically distinct retinal degenerative diseases, which are collectively referred to as the "bestrophinopathies". These five associated diseases are: Best vitelliform macular dystrophy, autosomal recessive bestrophinopathy, adult-onset vitelliform macular dystrophy, autosomal dominant vitreoretinochoroidopathy, and retinitis pigmentosa. The most common of these is Best vitelliform macular dystrophy. Bestrophin 1 (Best1), the protein encoded by the gene BEST1, has been the subject of a great deal of research since it was first identified nearly two decades ago. Today we know that Best1 functions as both a pentameric anion channel and a regulator of intracellular Ca2+ signaling. Best1 is an integral membrane protein which, within the eye, is uniquely expressed in the retinal pigment epithelium where it predominantly localizes to the basolateral plasma membrane. Within the brain, Best1 expression has been documented in both glial cells and astrocytes where it functions in both tonic GABA release and glutamate transport. The crystal structure of Best1 has revealed critical information about how Best1 functions as an ion channel and how Ca2+ regulates that function. Studies using animal models have led to critical insights into the physiological roles of Best1 and advances in stem cell technology have allowed for the development of patient-derived, "disease in a dish" models. In this article we review our knowledge of Best1 and discuss prospects for near-term clinical trials to test therapies for the bestrophinopathies, a currently incurable and untreatable set of diseases.
Assuntos
Bestrofinas/genética , DNA/genética , Mutação , Doenças Retinianas/genética , Animais , Bestrofinas/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Humanos , Camundongos , Doenças Retinianas/metabolismoRESUMO
The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner.
Assuntos
Baculoviridae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Animais , Baculoviridae/genética , Clonagem Molecular , Expressão Gênica , Ensaios de Triagem em Larga Escala , Células Sf9 , TransfecçãoRESUMO
Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here we report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.
Assuntos
Bacillus cereus/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Isoquinolinas/metabolismo , Ligantes , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Protoporfirinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Triptofano/análiseRESUMO
Human bestrophin-1 (hBest1) is a calcium-activated chloride channel from the retinal pigment epithelium, where mutations are associated with vitelliform macular degeneration, or Best disease. We describe the structure of a bacterial homolog (KpBest) of hBest1 and functional characterizations of both channels. KpBest is a pentamer that forms a five-helix transmembrane pore, closed by three rings of conserved hydrophobic residues, and has a cytoplasmic cavern with a restricted exit. From electrophysiological analysis of structure-inspired mutations in KpBest and hBest1, we find a sensitive control of ion selectivity in the bestrophins, including reversal of anion/cation selectivity, and dramatic activation by mutations at the cytoplasmic exit. A homology model of hBest1 shows the locations of disease-causing mutations and suggests possible roles in regulation.
Assuntos
Proteínas de Bactérias/química , Canais de Cloreto/química , Proteínas do Olho/química , Klebsiella pneumoniae , Bestrofinas , Cristalografia por Raios X , Condutividade Elétrica , Humanos , Conformação Proteica , Eletricidade EstáticaRESUMO
Microbial lipids activate T cells by binding directly to CD1 and T cell receptors (TCRs) or by indirect effects on antigen-presenting cells involving induction of lipid autoantigens, CD1 transcription, or cytokine release. To distinguish among direct and indirect mechanisms, we developed fluorescent human CD1b tetramers and measured T cell staining. CD1b tetramer staining of T cells requires glucose monomycolate (GMM) antigens, is specific for TCR structure, and is blocked by a recombinant clonotypic TCR comprised of TRAV17 and TRBV4-1, proving that CD1b-glycolipid complexes bind the TCR. GMM-loaded tetramers brightly stain a small subpopulation of blood-derived cells from humans infected with Mycobacterium tuberculosis, providing direct detection of a CD1b-reactive T cell repertoire. Polyclonal T cells from patients sorted with tetramers are activated by GMM antigens presented by CD1b. Whereas prior studies emphasized CD8(+) and CD4(-)CD8(-) CD1b-restricted clones, CD1b tetramer-based studies show that nearly all cells express the CD4 co-receptor. These findings prove a cognate mechanism whereby CD1b-glycolipid complexes bind to TCRs. CD1b tetramers detect a natural CD1b-restricted T cell repertoire ex vivo with unexpected features, opening a new investigative path to study the human CD1 system.