RESUMEN
We recently reported that the accumulation of myeloid-derived suppressor cells (MDSC), defined as CD33+HLA-DR-Lin-, has a direct role in the pathogenesis of myelodysplastic syndrome (MDS). In particular, CD33 is strongly expressed in MDSC isolated from patients with MDS where it has an important role in MDSC-mediated hematopoietic suppressive function through its activation by S100A9. Therefore, we tested whether blocking this interaction with a fully human, Fc-engineered monoclonal antibody against CD33 (BI 836858) suppresses CD33-mediated signal transduction and improves the bone marrow microenvironment in MDS. We observed that BI 836858 can reduce MDSC by antibody-dependent cellular cytotoxicity, which correlated with increases in granule mobilization and cell death. BI 836858 can also block CD33 downstream signaling preventing immune-suppressive cytokine secretion, which correlates with a significant increase in the formation of CFU-GM and BFU-E colonies. Activation of the CD33 pathway can cause reactive oxygen species (ROS)-induced genomic instability but BI 836858 reduced both ROS and the levels of double strand breaks and adducts (measured by comet assay and γH2AX). This work provides the ground for the development of a novel group of therapies for MDS aimed at MDSC and their disease-promoting properties with the goal of improving hematopoiesis in patients.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hematopoyesis/efectos de los fármacos , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Terapia Molecular Dirigida , Síndromes Mielodisplásicos/terapia , Células Supresoras de Origen Mieloide/efectos de los fármacos , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Médula Ósea/patología , Femenino , Ingeniería Genética , Inestabilidad Genómica , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/patología , Células Supresoras de Origen Mieloide/inmunología , Especies Reactivas de Oxígeno , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Nicho de Células MadreRESUMEN
Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with myelodysplastic syndrome (MDS) and chromosome 5q deletion (del(5q)). Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger degradation of mouse double minute 2 protein (MDM2), with consequent p53 transactivation. Herein we show that p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. More importantly, we show that lenalidomide (Len) acts to stabilize MDM2, thereby accelerating p53 degradation. Biochemical and molecular analyses showed that Len inhibits the haplodeficient protein phosphatase 2A catalytic domain alpha (PP2Acα) phosphatase resulting in hyperphosphorylation of inhibitory serine-166 and serine-186 residues on MDM2, and displaces binding of RPS14 to suppress MDM2 autoubiquitination whereas PP2Acα overexpression promotes drug resistance. Bone marrow specimens from del(5q) MDS patients resistant to Len overexpressed PP2Acα accompanied by restored accumulation of p53 in erythroid precursors. Our findings indicate that Len restores MDM2 functionality in the 5q- syndrome to overcome p53 activation in response to nucleolar stress, and therefore may warrant investigation in other disorders of ribosomal biogenesis.
Asunto(s)
Cromosomas Humanos Par 5 , Síndromes Mielodisplásicos/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Talidomida/análogos & derivados , Proteína p53 Supresora de Tumor/metabolismo , Animales , Deleción Cromosómica , Humanos , Lenalidomida , Ratones , Talidomida/farmacología , UbiquitinaciónRESUMEN
Our previous studies showed that high levels of soluble CD25 (sCD25) in the serum of patients with hepatocellular carcinoma (HCC) correlated with blunted effector T-cells (Teff) responses, tumour burden and poor survival. Understanding the interactions between Teff, CD4+CD25+ regulatory T cells (Treg) and soluble factors can identify novel therapeutic targets. In this study, we characterize the mechanisms by which HCC serum and sCD25 mediate suppression of Teff and evaluate the effect of sCD25 on the suppression assays with normal healthy control cells (NHC) at a 1:1 Treg to Teff cell ratio to determine whether sCD25 has any impact on Treg suppression. HCC serum and sCD25 suppressed Teff proliferation and downregulated CD25 expression on HCC Teff in a dose-dependent fashion with sCD25 doses above 3000 pg/ml. Treg from HCC and cirrhosis patients suppressed proliferation of target CD4+CD25- Teff in serum-free medium (SFM). HCC Treg showed a higher degree of suppression than cirrhosis-derived Treg. In contrast, Treg from NHC did not suppress target Teff in SFM. However, isolated Treg from all three study subjects (HCC, cirrhosis and NHC) suppressed CD4+CD25- Teff in serum conditions or in the presence of sCD25 in the range 6000-12,000 pg/ml. In conclusion, downregulation of CD25 cell surface expression on Teff is part of the overall suppressive mechanism of sCD25 and HCC serum on Teff responses. The observed sCD25 and HCC serum-mediated suppression is further influenced via novel immune-inhibitory interaction between CD4+CD25+ Treg and sCD25.
Asunto(s)
Carcinoma Hepatocelular/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Neoplasias Hepáticas/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Citometría de Flujo , Humanos , Subunidad alfa del Receptor de Interleucina-2/sangre , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/patología , Solubilidad , Linfocitos T/citología , Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
This study is to examine the monocyte-derived dendritic cell (DC) response to hepatitis C virus (HCV) in a cell culture system. Adherence-derived DCs were incubated with various titres of JFH-1 (HCV genotype 2a), generated from transfected Huh 7.5 cells or co-incubated with Newcastle disease virus (NDV). Infection and the type 1 interferon (IFN) response were assessed by real-time reverse transcriptase-polymerase chain reaction, morphology by light microscopy and immunophenotype by flow cytometry. Our data demonstrated no viral replication or particle release from DC after HCV infection. Morphologically, monocytes showed a tendency to shift to immature DCs when cultured with HCV, when compared with control monocytes. This shift was confirmed by flow cytometry and appeared to be related to viral titres. There was also an increase in immature DC numbers. HCV infection induced IFNß expression in DCs, and the amount seemed to be inversely correlated with viral titres indicating that HCV has the capacity to negatively regulate such cells. However, IFNα does not appear to be affected by direct contact with the virus. A strong IFNß signal induced by NDV in DC was substantially diminished by HCV. HCV negatively affects the maturation of DCs and suppresses the type 1 IFN response of DC. Our results suggest a mechanism of viral evasion of host immunity.