RESUMEN
Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Escape del Tumor/efectos de los fármacos , Animales , Antígeno B7-H1/análisis , Antígeno B7-H1/fisiología , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Ratones , Receptor de Muerte Celular Programada 1/análisis , Receptor de Muerte Celular Programada 1/fisiología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/análisisRESUMEN
Many potent nonsteroidal antiinflammatory drugs (NSAIDs) exert their effects by inhibiting the cyclooxygenase activity of prostaglandin H synthase-1 (PGHS1, thus disrupting prostaglandin biosynthesis. However, these drugs do not block the activation of NF-kappa B, an inducible transcription factor which regulates numerous inflammation-related genes. Here we demonstrate that PGHS1 peroxidase, a NSAID-insensitive activity of PGHS1, mediates NF-kappa B activation through an intracellular reactive oxygen signaling pathway. Overexpression of PGHS1 strongly potentiated NF-kappa B activation by phorbol esters and dramatically elevated the generation of intracellular reactive oxygen species (ROS) in response to low concentrations of t-butyl peroxide. Both functions were dependent on PGHS1 peroxidase activity and could be suppressed by the potent antioxidant pyrrolidine dithiocarbamate. In contrast, elimination of PGHS1 cyclooxygenase activity by NSAIDs or site-directed mutagenesis failed to block ROS production or NF-kappa B activation. Thus, PGHS1 peroxidase serves an intracellular signaling function leading to NF-kappa B activation, separable from its role in prostaglandin synthesis.
Asunto(s)
FN-kappa B/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Transducción de Señal , Antiinflamatorios no Esteroideos/farmacología , Secuencia de Bases , Línea Celular , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Especies Reactivas de Oxígeno/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Línea Celular , Fibroblastos/metabolismo , Factor 1 Regulador del Interferón , Ratones , Sondas Moleculares/genética , Datos de Secuencia Molecular , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/genéticaRESUMEN
Stimulation of the human monocytic cell line Mono Mac 6 with lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 microgram/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolerance operates at or upstream of the transcription level. The CD14 LPS receptor is, however, up-regulated (not down-regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional up-regulation of p50. Reporter gene analysis reveals that the NF-kappa B complex mobilized in tolerant cells is functionally inactive in that NF-kappa B-dependent luciferase constructs containing the human immunodeficiency virus long terminal repeat or the TNF 5'-region show only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimulation. The tolerant blood monocytes also up-regulate CD14, and they mobilize NF-kappa B with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the NF-kappa B complex.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Lipopolisacáridos/toxicidad , FN-kappa B/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/química , Tolerancia a Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Receptores de Lipopolisacáridos , Datos de Secuencia Molecular , Peso Molecular , Monocitos/efectos de los fármacos , FN-kappa B/química , ARN Mensajero/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia ArribaRESUMEN
In this study we report the identification of a novel transcription factor, termed Nuclear Factor-jun (NF-jun). This factor contributes to inducible transcription of the c-jun gene in human myeloid leukemia cells. NF-jun was, however, undetectable in nuclear proteins from human monocytes, granulocytes, resting T lymphocytes and lung fibroblasts. NF-jun shares several features with the well characterized NF-kappa B in that binding activity can be generated in cytosolic extracts by treatment with dissociating agents. In addition, binding of NF-jun to its recognition site is enhanced by treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, tumor necrosis factor alpha or the protein synthesis inhibitor cycloheximide (CHX). However, as revealed by competition assays and electrophoretic mobility shift assays, purified NF-kappa B fails to bind to the c-jun fragment which contains the NF-jun site, and this fragment fails to compete with NF-kappa B for binding. UV crosslinking showed that NF-jun contains a 55 and a 125 kDa protein species. These findings demonstrate that the c-jun gene can be regulated by a transcription factor distinct from AP-1. Our findings also indicate that while NF-jun has several features in common with the NF-kappa B binding protein including its subcellular localization and its ability to translocate from the cytoplasm to the nucleus, this factor recognizes a unique DNA sequence. Moreover, the activity of this protein is differentially regulated in various cell types. NF-jun might function as a signal transducing molecule in order to mediate rapid induction of the early response gene c-jun in a cell type- and stimulus-specific manner.