RESUMEN
The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Serina-Treonina Quinasas , Ratones , Humanos , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citocinas , Inflamación/tratamiento farmacológico , Isoformas de Proteínas , Antiinflamatorios/farmacología , Inmunidad Innata , Factores de TranscripciónRESUMEN
The mechanisms of HLA-DM-catalyzed peptide exchange remain uncertain. Here we found that all stages of the interaction of HLA-DM with HLA-DR were dependent on the occupancy state of the peptide-binding groove. High-affinity peptides were protected from removal by HLA-DM through two mechanisms: peptide binding induced the dissociation of a long-lived complex of empty HLA-DR and HLA-DM, and high-affinity HLA-DR-peptide complexes bound HLA-DM only very slowly. Nonbinding covalent HLA-DR-peptide complexes were converted into efficient HLA-DM binders after truncation of an N-terminal peptide segment that emptied the P1 pocket and disrupted conserved hydrogen bonds to HLA-DR. HLA-DM thus binds only to HLA-DR conformers in which a critical part of the binding site is already vacant because of spontaneous peptide motion.
Asunto(s)
Antígenos HLA-D/metabolismo , Antígeno HLA-DR2/metabolismo , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Presentación de Antígeno , Células CHO , Catálisis , Cricetinae , Cricetulus , Antígenos HLA-D/química , Antígenos HLA-D/genética , Antígeno HLA-DR2/química , Antígeno HLA-DR2/genética , Humanos , Modelos Químicos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Unión Proteica , Resonancia por Plasmón de Superficie , Transgenes/genéticaRESUMEN
Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Inmunidad Humoral/fisiología , Memoria Inmunológica/fisiología , Modelos Inmunológicos , Proteínas Tirosina Quinasas/inmunología , Agammaglobulinemia Tirosina Quinasa , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Humanos , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genéticaRESUMEN
Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.
Asunto(s)
Linfocitos B/inmunología , Glomerulonefritis/inmunología , Lupus Eritematoso Sistémico/inmunología , Piperidinas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/uso terapéutico , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Centro Germinal/citología , Glomerulonefritis/metabolismo , Glomerulonefritis/prevención & control , Riñón/inmunología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/prevención & control , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NZB , Piperidinas/farmacología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Pirazoles/farmacología , Receptores Fc , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Regulación Alostérica , Sitio Alostérico , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two ß strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.
Asunto(s)
Mutación , N-Acetilglucosaminiltransferasas/genética , Oligosacáridos/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Células Clonales , Cricetinae , Glicosilación , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Oligosacáridos/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
OBJECTIVES: To characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys. METHODS: Direct binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes. RESULTS: 2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes. CONCLUSIONS: SMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD20/efectos de los fármacos , Antígenos CD20/inmunología , Anticuerpos de Cadena Única/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Factores Inmunológicos/farmacología , Técnicas In Vitro , Modelos Lineales , Macaca fascicularis , Distribución Aleatoria , Rituximab , Sensibilidad y EspecificidadRESUMEN
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Galectina 1/fisiología , Hipoglucemiantes/administración & dosificación , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/prevención & control , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/prevención & control , Femenino , Galectina 1/administración & dosificación , Humanos , Inyecciones Intraperitoneales , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Ratones TransgénicosRESUMEN
Rapid binding of peptides to MHC class II molecules is normally limited to a deep endosomal compartment where the coordinate action of low pH and HLA-DM displaces the invariant chain remnant CLIP or other peptides from the binding site. Exogenously added peptides are subject to proteolytic degradation for extended periods of time before they reach the relevant endosomal compartment, which limits the efficacy of peptide-based vaccines and therapeutics. In this study, we describe a family of small molecules that substantially accelerate the rate of peptide binding to HLA-DR molecules in the absence of HLA-DM. A structure-activity relationship study resulted in analogs with significantly higher potency and also defined key structural features required for activity. These compounds are active over a broad pH range and thus enable efficient peptide loading at the cell surface. The small molecules not only enhance peptide presentation by APC in vitro, but are also active in vivo where they substantially increase the fraction of APC on which displayed peptide is detectable. We propose that the small molecule quickly reaches draining lymph nodes along with the coadministered peptide and induces rapid loading of peptide before it is destroyed by proteases. Such compounds may be useful for enhancing the efficacy of peptide-based vaccines and other therapeutics that require binding to MHC class II molecules.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos HLA-DR/inmunología , Péptidos/química , Péptidos/inmunología , Animales , Antígenos HLA-D/inmunología , Antígenos HLA-D/metabolismo , Antígenos HLA-DR/metabolismo , Ratones , Ratones Transgénicos , Relación Estructura-ActividadRESUMEN
The inhibitory receptor programmed death-1 (PD-1) is present on CD8(+) T cells in chronic hepatitis C virus (HCV), but expression patterns in spontaneously resolving infections are incompletely characterized. Here we report that PD-1 was usually absent on memory CD8(+) T cells from chimpanzees with resolved infections, but sustained low-level expression was sometimes observed in the absence of apparent virus replication. PD-1-positive memory T cells expanded and displayed antiviral activity upon reinfection with HCV, indicating conserved function. This animal model should facilitate studies of whether PD-1 differentially influences effector and memory T-cell function in resolved versus persistent human infections.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/biosíntesis , Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Animales , Citometría de Flujo , Pan troglodytesRESUMEN
The autoimmune disease type 1 diabetes in humans and NOD mice is determined by multiple genetic factors, among the strongest of which is the inheritance of diabetes-permissive MHC class II alleles associated with susceptibility to disease. Here we examined whether expression of MHC class II alleles associated with resistance to disease could be used to prevent the occurrence of diabetes. Expression of diabetes-resistant MHC class II I-Abeta chain molecules in NOD mice following retroviral transduction of autologous bone marrow hematopoietic stem cells prevented the development of autoreactive T cells by intrathymic deletion and protected the mice from the development of insulitis and diabetes. These data suggest that type 1 diabetes could be prevented in individuals expressing MHC alleles associated with susceptibility to disease by restoration of protective MHC class II expression through genetic engineering of hematopoietic stem cells.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética , Transducción Genética , Animales , Glucemia/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/inmunología , Susceptibilidad a Enfermedades , Femenino , Genes MHC Clase II , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Linfocitos T/inmunología , Linfocitos T/fisiologíaRESUMEN
Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos HLA-DR/inmunología , Hepacivirus/inmunología , Memoria Inmunológica , Animales , Linfocitos T CD4-Positivos/metabolismo , Genes MHC Clase II , Hepatitis C/inmunología , Humanos , Péptidos/química , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Viremia/inmunologíaRESUMEN
The identification of new tumor-associated antigens (TAA) is critical for the development of effective immunotherapeutic strategies, particularly in diseases like B-cell acute lymphoblastic leukemia (B-ALL), where few target epitopes are known. To accelerate the identification of novel TAA in B-ALL, we used a combination of expression profiling and reverse immunology. We compared gene expression profiles of primary B-ALL cells with their normal counterparts, B-cell precursors. Genes differentially expressed by B-ALL cells included many previously identified as TAA in other malignancies. Within this set of overexpressed genes, we focused on those that may be functionally important to the cancer cell. The apoptosis-related molecule, BAX, was highly correlated with the ALL class distinction. Therefore, we evaluated BAX and its isoforms as potential TAA. Peptides from the isoform BAX-delta bound with high affinity to HLA-A*0201 and HLA-DR1. CD8+ CTLs specific for BAX-delta epitopes or their heteroclitic peptides could be expanded from normal donors. BAX-delta-specific T cells lysed peptide-pulsed targets and BAX-delta-expressing leukemia cells in a MHC-restricted fashion. Moreover, primary B-ALL cells were recognized by BAX-delta-specific CTL, indicating that this antigen is naturally processed and presented by tumor cells. This study suggests that (a) BAX-delta may serve as a widely expressed TAA in B-ALL and (b) gene expression profiling can be a generalizable tool to identify immunologic targets for cancer immunotherapy.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/inmunología , Adolescente , Secuencia de Aminoácidos , Antígenos de Neoplasias/biosíntesis , Linfocitos B/inmunología , Línea Celular Tumoral , Niño , Preescolar , Epítopos de Linfocito T/inmunología , Perfilación de la Expresión Génica , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Antígenos HLA-DR/inmunología , Humanos , Lactante , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Linfocitos T Citotóxicos/inmunología , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
Asunto(s)
Interleucina-2/farmacología , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Linfocitos T/enzimología , Diseño de Fármacos , Semivida , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Recognition of self-peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR-pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Sinapsis Inmunológicas/inmunología , Esclerosis Múltiple/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Movimiento Celular/inmunología , Antígenos HLA/inmunología , Humanos , Immunoblotting , Molécula 1 de Adhesión Intercelular/inmunología , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , FosforilaciónRESUMEN
Peptide loading of MHC class II (MHCII) molecules is directly catalyzed by the MHCII-like molecule HLA-DM (DM). Another MHCII-like molecule, HLA-DO (DO), associates with DM, thereby modulating DM function. The biological role of DO-mediated regulation of DM activity in vivo remains unknown; however, it has been postulated that DO expression dampens presentation of self antigens, thereby preventing inappropriate T cell activation that ultimately leads to autoimmunity. To test the idea that DO modulation of the MHCII self-peptide repertoire mediates self tolerance, we generated NOD mice that constitutively overexpressed DO in DCs (referred to herein as NOD.DO mice). NOD mice are a mouse model for type 1 diabetes, an autoimmune disease mediated by the destruction of insulin-secreting pancreatic beta cells. Our studies showed that diabetes development was completely blocked in NOD.DO mice. Similar to NOD mice, NOD.DO animals selected a diabetogenic T cell repertoire, and the numbers and function of Tregs were normal. Indeed, immune system function in NOD.DO mice was equivalent to that in NOD mice. NOD.DO DCs, however, presented an altered MHCII-bound self-peptide repertoire, thereby preventing the activation of diabetogenic T cells and subsequent diabetes development. These studies show that DO expression can shape the overall MHCII self-peptide repertoire to promote T cell tolerance.
Asunto(s)
Presentación de Antígeno , Autoantígenos/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Inmunocompetencia , Animales , Antígeno CD11c/fisiología , Diabetes Mellitus Tipo 1/inmunología , Femenino , Antígenos HLA-D/fisiología , Antígenos de Histocompatibilidad Clase II/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T Reguladores/inmunologíaRESUMEN
HLA-DRB1*0101 is associated with susceptibility to human T lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Here, we used a synthetic tetramer of DRB1*0101 and its epitope peptide to analyze HTLV-1-specific CD4(+) T cells ex vivo. The frequency of tetramer(+)CD4(+) T cells was significantly greater in patients with HAM/TSP than in healthy HTLV-1 carriers (HCs) at a given proviral load and correlated with HTLV-1 tax messenger RNA expression in HCs but not in patients with HAM/TSP. These cells displayed an early to intermediate effector memory phenotype and were preferentially infected by HTLV-1. T cell receptor gene analyses of 2 unrelated DRB1*0101-positive patients with HAM/TSP showed similar Vbeta repertoires and amino acid motifs in complementarity-determining region 3. Our data suggest that efficient clonal expansion of virus-specific CD4(+) T cells in patients with HAM/TSP does not simply reflect higher viral burden but rather reflects a rapid turnover caused by preferential infection and/or in vivo stimulation by major histocompatibility complex-peptide complexes.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos HLA-A/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Epítopos Inmunodominantes/inmunología , Paraparesia Espástica Tropical/inmunología , Alelos , Secuencia de Aminoácidos , Femenino , Genes pX/genética , Genes pX/inmunología , Predisposición Genética a la Enfermedad , Antígenos HLA-A/genética , Cadenas HLA-DRB1 , Antígenos HTLV-I/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Paraparesia Espástica Tropical/genética , Paraparesia Espástica Tropical/virología , Fenotipo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T/inmunología , Carga ViralRESUMEN
Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.
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Toxinas Bacterianas/química , Enterotoxinas/química , Epítopos/genética , Modelos Moleculares , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Transducción de Señal/inmunología , Superantígenos/química , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Toxinas Bacterianas/metabolismo , Cristalografía , Enterotoxinas/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Superantígenos/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. Large lateral surfaces involved in the DM:HLA-DR (DR) interaction have been defined, but the mechanism of catalysis is not understood. In this study, we describe four small molecules that accelerate DM-catalyzed peptide exchange. Mechanistic studies demonstrate that these small molecules substantially enhance the catalytic efficiency of DM, indicating that they make the transition state of the DM:DR/peptide complex energetically more favorable. These compounds fall into two functional classes: two compounds are active only in the presence of DM, and binding data for one show a direct interaction with DM. The remaining two compounds have partial activity in the absence of DM, suggesting that they may act at the interface between DM and DR/peptide. A hydrophobic ridge in the DMbeta1 domain was implicated in the catalysis of peptide exchange because the activity of three of these enhancers was substantially reduced by point mutations in this area.
Asunto(s)
Antígenos HLA-D/metabolismo , Péptidos/farmacología , Sitios de Unión , Catálisis/efectos de los fármacos , Antígenos HLA-D/química , Antígenos HLA-D/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación/genética , Péptidos/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-gamma-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7-CD45RA-) and functional properties (IFN-gamma+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.