RESUMEN
Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
Asunto(s)
Anticuerpos , Especificidad de Anticuerpos , Proteínas de la Membrana , Proteolisis , Ubiquitina-Proteína Ligasas , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Neoplasias Colorrectales/metabolismo , Ligandos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: Over the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding. RESULTS: We tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals. CONCLUSIONS: Unlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway.
Asunto(s)
Asma , Degeneración Macular , Bioensayo , Biomarcadores , Desarrollo de Medicamentos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Interleucina-33 , Sensibilidad y EspecificidadRESUMEN
Rabbits produce robust antibody responses and have unique features in their antibody repertoire that make them an attractive alternative to rodents for in vivo discovery. However, the frequent occurrence of a non-canonical disulfide bond between complementarity-determining region (CDR) H1 (C35a) and CDRH2 (C50) is often seen as a liability for therapeutic antibody development, despite limited reports of its effect on antibody binding, function, and stability. Here, we describe the discovery and humanization of a human-mouse cross-reactive anti-programmed cell death 1 (PD-1) monoclonal rabbit antibody, termed h1340.CC, which possesses this non-canonical disulfide bond. Initial removal of the non-canonical disulfide resulted in a loss of PD-1 affinity and cross-reactivity, which led us to explore protein engineering approaches to recover these. First, guided by the sequence of a related clone and the crystal structure of h1340.CC in complex with PD-1, we generated variant h1340.SA.LV with a potency and cross-reactivity similar to h1340.CC, but only partially recovered affinity. Side-by-side developability assessment of both h1340.CC and h1340.SA.LV indicate that they possess similar, favorable properties. Next, and prompted by recent developments in machine learning (ML)-guided protein engineering, we used an unbiased ML- and structure-guided approach to rapidly and efficiently generate a different variant with recovered affinity. Our case study thus indicates that, while the non-canonical inter-CDR disulfide bond found in rabbit antibodies does not necessarily constitute an obstacle to therapeutic antibody development, combining structure- and ML-guided approaches can provide a fast and efficient way to improve antibody properties and remove potential liabilities.
Asunto(s)
Anticuerpos , Receptor de Muerte Celular Programada 1 , Conejos , Animales , Ratones , Humanos , Regiones Determinantes de Complementariedad/química , Ingeniería de Proteínas/métodosRESUMEN
Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Modelos Moleculares , Receptores de Complemento/genética , Animales , Artritis Experimental/complicaciones , Resorción Ósea/etiología , Inactivadores del Complemento , Cristalización , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Ratones , Receptores de Complemento/químicaRESUMEN
CRIg is a recently discovered complement C3 receptor expressed on a subpopulation of tissue-resident macrophages. The extracellular IgV domain of CRIg (CRIg-ECD) holds considerable promise as a potential therapeutic because it selectively inhibits the alternative pathway of complement by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binds weakly to the convertase subunit C3b (K(D) = 1.1 microm), and thus a relatively high concentration of protein is required to reach nearly complete complement inhibition. To improve therapeutic efficacy while minimizing risk of immunogenicity, we devised a phage display strategy to evolve a high affinity CRIg-ECD variant with a minimal number of mutations. Using the crystal structure of CRIg in complex with C3b as a guide for library design, we isolated a CRIg-ECD double mutant (Q64R/M86Y, CRIg-v27) that showed increased binding affinity and improved complement inhibitory activity relative to CRIg-ECD. In a mouse model of arthritis, treatment with a Fc fusion of CRIg-v27 resulted in a significant reduction in clinical scores compared with treatment with an Fc fusion of CRIg-ECD. This study clearly illustrates how phage display technology and structural information can be combined to generate proteins with nearly natural sequences that act as potent complement inhibitors with greatly improved therapeutic efficacy.
Asunto(s)
Artritis/tratamiento farmacológico , Receptores de Complemento 3b/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Sustitución de Aminoácidos , Animales , Artritis/metabolismo , Complemento C3b/genética , Complemento C3b/metabolismo , Complemento C5/genética , Complemento C5/metabolismo , Vía Alternativa del Complemento/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación Missense , Estructura Terciaria de Proteína/fisiología , Conejos , Receptores de Complemento 3b/química , Receptores de Complemento 3b/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Relación Estructura-ActividadRESUMEN
An important function of the complement cascade is to coat self and foreign particles with C3-proteins that serve as ligands for phagocytic receptors. Although tissue resident macrophages play an important role in complement-mediated clearance, the receptors coordinating this process have not been well characterized. In the present study, we identified a subpopulation of resident peritoneal macrophages characterized by high expression of complement receptor of the Ig superfamily (CRIg), a recently discovered complement C3 receptor. Macrophages expressing CRIg showed significantly increased binding and subsequent internalization of complement-opsonized particles compared with CRIg negative macrophages. CRIg internalized monovalent ligands and was able to bind complement-opsonized targets in the absence of Ca(2+) and Mg(2+), which differs from the beta(2)-integrin CR3 that requires divalent cations and polyvalent ligands for activation of the receptor. Although CRIg dominated in immediate binding of complement-coated particles, CRIg and CR3 contributed independently to subsequent particle phagocytosis. CRIg thus identifies a subset of tissue resident macrophages capable of increased phagocytosis of complement C3-coated particles, a function critical for immune clearance.
Asunto(s)
Complemento C3/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Receptores de Complemento/inmunología , Animales , Antígenos CD18/inmunología , Calcio/inmunología , Regulación de la Expresión Génica/inmunología , Ligandos , Magnesio/inmunología , Ratones , Ratones Endogámicos AKR , Ratones Noqueados , Receptores de Complemento/agonistasRESUMEN
Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.
Asunto(s)
Mastocitos/enzimología , Serina Proteasas/metabolismo , Traslado Adoptivo , Animales , Complejo Antígeno-Anticuerpo , Regulación Enzimológica de la Expresión Génica , Histamina/metabolismo , Ratones , Ratones Noqueados , Neutrófilos , Serina Proteasas/genética , Serotonina/metabolismoRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.
Asunto(s)
Activación de Complemento/genética , Atrofia Geográfica/fisiopatología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Atrofia/patología , Activación de Complemento/fisiología , Complemento C3/genética , Complemento C3/fisiología , Complemento C4/genética , Complemento C4/fisiología , Atrofia Geográfica/genética , Humanos , Degeneración Macular/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33(+) Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.
Asunto(s)
Inmunidad Innata , Interleucina-33/metabolismo , Degeneración Macular/inmunología , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Núcleo Celular/inmunología , Citocinas/metabolismo , Células Ependimogliales/inmunología , Células Ependimogliales/patología , Femenino , Humanos , Técnicas In Vitro , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33/química , Interleucina-33/deficiencia , Interleucina-33/genética , Mácula Lútea/inmunología , Mácula Lútea/patología , Degeneración Macular/genética , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Ratas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
IFN-gamma induced transcription of class II transactivator (CIITA), a major regulator of MHC class II gene expression, is directed by the CIITA type IV promoter. The IFN-gamma activation of the CIITA type IV promoter is mediated by STAT1 and IRF-1, which bind to the GAS and IRF-E of the promoter, respectively. We and others have determined that IRF-2, another member of the IRF family, also activates the CIITA type IV promoter, by binding to the IRF-E. Also, IRF-2 cooperates with IRF-1 to activate the promoter. DNA binding analyses determined that IRF-1 and IRF-2 can co-occupy the IRF-E of the CIITA type IV promoter. To further understand the mechanism of IRF-1 and IRF-2 cooperativity in the activation of CIITA type IV promoter, we characterized the binding of IRF-1 and IRF-2 to the CIITA IRF-E and mapped the domains of IRF-2 required for the cooperative transactivation. Off-rate experiments revealed that the IRF-2/IRF-E complex was more stable than the IRF-1/IRF-E complex and that the affinity of IRF-1 for the IRF-E was increased when IRF-1 co-occupied the IRF-E with IRF-2. Deletion analysis of functional domains of IRF-2 revealed that a previously described latent activation domain of IRF-2 was essential for IRF-2 transactivation and participated in cooperative activation of the CIITA promoter by IRF-1 and IRF-2. However, the DNA binding domain of IRF-2 was sufficient for cooperativity with IRF-1 in the activation of the CIITA type IV promoter. DNA binding assay demonstrated that, like the full-length IRF-2, the IRF-2 DNA binding domain could co-occupy the CIITA IRF-E with IRF-1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica/genética , Proteínas Nucleares , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras , Transactivadores/genética , Factores de Transcripción , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Factor 1 Regulador del Interferón , Factor 2 Regulador del Interferón , Interferón gamma/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Multiple sclerosis and its preclinical model, experimental autoimmune encephalomyelitis, are marked by perivascular inflammation and demyelination. Myeloid cells, derived from circulating progenitors, are a prominent component of the inflammatory infiltrate and are believed to directly contribute to demyelination and axonal damage. How the cytotoxic activity of these myeloid cells is regulated is poorly understood. We identify CMRF-35-like molecule-1 (CLM-1) as a negative regulator of autoimmune demyelination. CLM-1 is expressed on inflammatory myeloid cells present in demyelinating areas of the spinal cord after immunization of mice with MOG35-55 (myelin oligodendrocyte glycoprotein) peptide. Absence of CLM-1 resulted in significantly increased nitric oxide and proinflammatory cytokine production, along with increased demyelination and worsened clinical scores, whereas T cell responses in the periphery or in the spinal cord remained unaffected. This study thus identifies CLM-1 as a negative regulator of myeloid effector cells in autoimmune demyelination.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Células Mieloides/inmunología , Receptores Inmunológicos/inmunología , Animales , Citocinas/genética , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Glicoproteínas/efectos adversos , Glicoproteínas/farmacología , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito , Células Mieloides/patología , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/farmacología , Receptores Inmunológicos/genéticaRESUMEN
Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.
Asunto(s)
Anticuerpos , Complemento C3b/inmunología , Vía Alternativa del Complemento/fisiología , Fragmentos Fab de Inmunoglobulinas , Conformación Proteica , Animales , Anticuerpos/química , Anticuerpos/metabolismo , C3 Convertasa de la Vía Alternativa del Complemento/metabolismo , Complemento C3b/química , Complemento C3b/genética , Complemento C3b/metabolismo , C5 Convertasa de la Vía Alternativa del Complemento/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Macaca mulatta , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Receptores de Complemento 3b/química , Receptores de Complemento 3b/metabolismoRESUMEN
The response of thymocytes to pre-T cell receptor (pre-TCR) signaling includes proliferation and gene rearrangement, two cellular processes that are incompatible. The control of proliferation by pre-TCR signals depends on the activities of the transcription factors RORgammat, Egr3, E12, and E47. Here, we describe a regulatory network in which interplay between these factors ensures transient proliferation that is temporally distinct from gene rearrangement. RORgammat expression was elevated after pre-TCR signaling, and RORgammat promoted gene rearrangement in CD4+, CD8+ cells by inhibiting cell division, promoting survival via Bcl-X(L), and inducing Rag2. Egr3 was transiently induced by pre-TCR signals and promoted a distinct proliferative phase by reducing E protein-dependent RORgammat expression and interacting with RORgammat to prevent induction of target genes. After Egr3 subsided, the expression and function of RORgammat increased. Thus, transient induction of Egr3 delays the effects of RORgammat and enables pre-TCR signaling to induce both proliferation and gene rearrangement.
Asunto(s)
Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Linfocitos T/inmunología , Factores de Transcripción TCF/metabolismo , Animales , Elementos E-Box , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Reordenamiento Génico de Linfocito T , Proteínas Inhibidoras de la Diferenciación/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Mutantes , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/genética , Ratas , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
In the absence of selection, CD4+, CD8+ double-positive (DP) thymocytes will die after 3-4 days. The mechanism for regulating the life span of DP cells is unknown. Previously, we demonstrated that the zinc finger transcription factor, early growth response gene 3 (Egr3), promotes proliferation during the transition from double negative (DN) to DP. In this study we demonstrate a novel role for Egr3 in controlling DP thymocyte survival in mice. Constitutive transgenic expression of Egr3 in thymocytes increases apoptosis among DP cells and shortens their survival in vitro. In addition, DP cells in Egr3 transgenic mice have poor expression of TCRalpha, and based on the predominant usage of 3' Valpha and 5' Jalpha gene segments, the low level of TCRalpha expression is a result of DP death soon after the initiation of TCRalpha rearrangements. Constitutive transgenic expression of Egr3 results in poor expression of Bcl-x(L) and the thymic isoform of retinoic acid receptor-related orphan receptor gamma (RORgammat) in DP thymocytes, two molecules that are required in DP cells for normal life span. Egr3 expression induced by pre-TCR signals in nontransgenic mice is transient and returns to background levels before RORgammat or Bcl-x(L) is induced. The data support a model in which Egr3 must be transiently induced in response to pre-TCR signals, so that the expression of the prosurvival molecules, RORgammat and Bcl-x(L), can be elevated only after the proliferative signal provided by Egr3 has subsided.
Asunto(s)
Antígenos CD4/análisis , Antígenos CD8/análisis , Proteínas de Unión al ADN/fisiología , Linfocitos T/fisiología , Factores de Transcripción/fisiología , Animales , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteína 3 de la Respuesta de Crecimiento Precoz , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Ratones , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/genética , Factores de Transcripción/genética , Proteína bcl-XRESUMEN
Responses to partial agonist TCR signals include positive selection of thymocytes, survival of naive T cells, and homeostatic proliferation. As part of an effort to understand the molecular basis of these processes, we have determined how agonist and partial agonist ligands act differently to induce a change in gene expression. We have found that the early growth response gene 1 (Egr1) promoter is activated by agonist and partial agonist ligands, but the partial agonist induces 10-fold lower promoter activity. Both agonist and partial agonist ligands require all six serum response elements in the Egr1 promoter to reach maximum induction. Although slightly fewer cells respond to the partial agonist, all of the responding cells have reduced activity compared with the cells responding to agonist. The factors binding to the serum response elements of the Egr1 promoter form a ternary complex (TC) consisting of serum response factor and either Elk-1 or serum response factor accessory protein-1a. Formation of a stable TC and inducible promoter activity are both dependent on extracellular signal-related kinase activation. Examination of TC formation over time reveals that this complex is induced well by partial agonist ligands, but it is not sustained, whereas agonist stimulation induces longer lived TCs. Therefore, the data suggest that both agonist and partial agonist ligands can induce formation of multiple TC on the Egr1 promoter, but the ability of the agonist ligand to maintain these complexes for an extended time results in the increased potency of the agonist.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/inmunología , Proteínas Inmediatas-Precoces , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas/inmunología , Receptores de Antígenos de Linfocitos T/agonistas , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Factores de Transcripción/genética , Animales , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta Inmunológica , Proteína 1 de la Respuesta de Crecimiento Precoz , Activación Enzimática/genética , Activación Enzimática/inmunología , Ligandos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Elemento de Respuesta al Suero/inmunología , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transducción de Señal/genética , Especificidad por Sustrato/genética , Especificidad por Sustrato/inmunología , Linfocitos T/enzimología , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteína Elk-1 con Dominio ets , Proteína Elk-4 del Dominio etsRESUMEN
In thymocytes developing in the alphabeta lineage, the transition from CD4, CD8 double negative (DN) to CD4, CD8 double positive (DP) is associated with several rounds of cell division and changes in the expression of multiple genes. This transition is induced by the formation of a pre-TCR that includes a rearranged TCR beta-chain and the pre-TCR alpha-chain. The mechanism by which the pre-TCR influences both gene expression and proliferation has not been defined. We have evaluated the role played by early growth response gene 3 (Egr3) in translating pre-TCR signals into differentiation and proliferation. Egr3 is a transcriptional regulator that contains a zinc-finger DNA binding domain. We find that Egr3-deficient mice have a reduced number of thymocytes compared with wild-type mice, and that this is due to poor proliferation during the DN to DP transition. Treatment of both Egr3(+/+) and Egr3(-/-) mice on the Rag1(-/-) background with anti-CD3epsilon Ab in vivo results in similar differentiation events, but reduced cell recovery in the Egr3(-/-) mice. We have also generated transgenic mice that express high levels of Egr3 constitutively, and when these mice are bred onto a Rag1(-/-) background they exhibit increased proliferation in the absence of stimulation and have pre-TCR alpha-chain and CD25 down-regulation, as well as increased Calpha expression. The results show that Egr3 is an important regulator of proliferation in response to pre-TCR signals, and that it also may regulate some specific aspects of differentiation.
Asunto(s)
Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Proteínas de Unión al ADN/fisiología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Factores de Transcripción/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , División Celular/genética , División Celular/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteína 3 de la Respuesta de Crecimiento Precoz , Regulación del Desarrollo de la Expresión Génica/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Precursores de Proteínas/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transgenes/inmunologíaRESUMEN
Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Antígenos de Histocompatibilidad/fisiología , Proteínas Inmediatas-Precoces , ARN Mensajero/metabolismo , Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Animales , Células Presentadoras de Antígenos/fisiología , Antígenos/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz , Complejo Mayor de Histocompatibilidad/fisiología , Ratones , Péptidos/metabolismoRESUMEN
Early growth response gene 1 (Egr1) codes for a transcriptional regulator that contains a zinc-finger DNA binding domain. Egr1 expression is induced by a variety of extracellular stimuli including TCR-ligand interactions. Its pattern of expression in the thymus and dependence on ERK activation have led to speculation that it has a role in T cell development, but the exact nature of this role has been undefined. To more clearly define the role of Egr1 in thymocyte development, we have analyzed thymocytes from Egr1-deficient mice. We find that thymuses from Egr1-deficient mice contain twice as many cells as age-matched controls, and the increase in thymocyte number is apparent at the early CD4/CD8 double negative stage of development. Subsequent maturation to the CD4/CD8 double positive stage and survival of the double positive cells both appear normal in Egr1-deficient animals. We also find that Egr1 promotes positive selection of both CD4 and CD8 single positive cells without playing a major role in negative selection. Egr1 influences positive selection by enhancing expression of the helix-loop-helix inhibitor Id3 and the anti-apoptosis molecule bcl-2. Thus, Egr1 translates developmental signals into appropriate changes in gene expression at multiple stages of thymocyte development.